Bernd Thiede

1. Koehler, C.J., Arntzen, M.Ø., de Souza, G.A., Thiede, B. (2013) An approach for triplex-isobaric peptide termini labeling (Triplex-IPTL). Anal. Chem., 85, 2478-2485.

2. Thiede, B., Koehler, C.J., Strozynski, M., Treumann, A., Stein, R., Zimny-Arndt, U., Schmid, M., Jungblut, P.R. (2013) High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture (SILAC), two-dimensional gel electrophoresis (2DE) and nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer. Mol. Cell. Proteomics, 12, 529-538.

3. Arntzen, M.A., Boddie, P., Frick, R., Koehler, C.J., Thiede, B. (2015) Consolidation of proteomics data in the Cancer proteomics database. Proteomics, 15, 3765-3771.

4. Trung, T.T., Strozynski, M.A., Thiede, B. (2017) Quantitative phosphoproteome analysis of cisplatin-induced apoptosis in Jurkat T cells, Proteomics, 17, 1600470.

5. Trung, T.T., Bollineni, R.C., Strozynski, M.A., Koehler, C.J., Thiede, B. (2017) Identification of alternative splice variants using unique tryptic peptide sequences for database searches, J. Proteome Res., 16, 2571-2578.

6. Bollineni, R.C., Koehler, C.J., Gislefoss, R.E., Anonsen, J.H., Thiede, B. (2018) Large-scale intact glycopeptide identification by Mascot database search. Sci. Rep. 8, 2117.

• Sargautis, Domantas & Thiede, Bernd (2026). LC-MS peptide mapping of monoclonal antibodies using the mirror proteases trypsin and Tryp-N. Journal of Pharmaceutical and Biomedical Analysis. ISSN 0731-7085. 272. doi: 10.1016/j.jpba.2026.117361. Fulltekst i vitenarkiv
• Iversen, Lars; Garrastacho, Manuel Ramirez; Hansen, Pernille May; Thiede, Bernd; Thyssen, Jacob Pontoppidan & Rasmussen, Marie Louise Roed [Vis alle 9 forfattere av denne artikkelen] (2026). Proteomic profiling of tear fluid in patients with atopic keratoconjunctivitis. The ocular surface. ISSN 1542-0124. 41, s. 66–75. doi: 10.1016/j.jtos.2026.05.005. Fulltekst i vitenarkiv
• Kärkkäinen, Virve; Saari, Toni; Rusanen, Minna; Uusitalo, Hannu; Leinonen, Ville & Thiede, Bernd [Vis alle 9 forfattere av denne artikkelen] (2026). Altered tear fluid protein expression in persons with mild cognitive impairment. Journal of Alzheimer's Disease. ISSN 1387-2877. 110(1), s. 109–119. doi: 10.1177/13872877251413291. Fulltekst i vitenarkiv
• Nguyen, Xuan Thang; Martiensen, Emily Louise Beecher; Abdelhalim, Mohamed; Tran, Trung; Ledsaak, Marit & Rogne, Marie [Vis alle 8 forfattere av denne artikkelen] (2026). Proteome-wide multi-omics profiling of osteosarcoma transcription factor networks. bioRxiv (preprint article). doi: 10.64898/2026.03.29.714917. Fulltekst i vitenarkiv Vis sammendrag

  Osteosarcoma (OS) is an aggressive bone cancer that most commonly affects children and young adults. OS exhibits a high degree of genomic complexity, as well as cellular plasticity, and dynamic transcriptional regulation is suggested to contribute to treatment resistance and metastasis. Cell lines are well characterized as models to advance our knowledge on OS biology. HOS and U2OS cells have increased invasiveness and higher migratory ability compared with MG63. In this study, we employed a tandem array of consensus transcription factor response elements (catTFREs) proteomic approach to characterize transcription factor (TF) regulatory networks related to OS aggressiveness. We mapped 7,594 proteins and enriched 352 transcription factors and coregulators. When we integrated proteomics with cell line specific gene expression and chromatin accessibility we classified the proteins into different OS cell line dependent sub-clusters and identified TFs and coregulators common for all cell lines and specific for individual cell lines. We demonstrate that RUNX2, MYBL2 and HMGA2 are specifically enriched in HOS and U2OS and may be linked to the cell aggressiveness. ETV5, JUNB, NFIX and ZEB1 were among TFs specific to MG63. Our analysis provides a more comprehensive understanding of the transcriptional drivers that shape OS regulatory landscapes and may have future therapeutic implications.

• Lauretis, Angela De; Wang, Qiang; Santacroce, Marco; Thiede, Bernd; Ma, Qianli & Lyngstadaas, Ståle Petter [Vis alle 9 forfattere av denne artikkelen] (2025). A Comparative Study of the Impact of Chemical Debridement Products on the Proteomic Profile of the Salivary Pellicle and Cell Adhesion on OsseoSpeed Titanium Dental Implant Surfaces. ACS Applied Materials & Interfaces. ISSN 1944-8244. 17(39), s. 54592–54592. doi: 10.1021/acsami.5c14570. Fulltekst i vitenarkiv
• Amono, Racheal; Rønning, Snöa Amanda Tora Nyx Fredlund; Chesnais, Morgane; Thiede, Bernd; Markussen, Turhan & Evensen, Øystein [Vis alle 7 forfattere av denne artikkelen] (2025). Defined domains and cleavage determine the diverse functions of piscine myocarditis virus p33 protein. Frontiers in Microbiology. 16. doi: 10.3389/fmicb.2025.1633241. Fulltekst i vitenarkiv Vis sammendrag

  Piscine myocarditis virus (PMCV) causes chronic, necrotizing myocarditis in Atlantic salmon. Originally, PMCV was identified based on its genetic homology and genomic organization, indicating a relationship to viruses of the Ghabrivirales order, specifically the former Totiviridae family, whose members predominantly infect fungi or protozoans and lack an extracellular life cycle stage. However, PMCV was the first virus of this order found to infect a vertebrate host. Since then, other piscine viruses and viruses infecting terrestrial and aquatic arthropods have been described and recently assigned to new virus families within the order. PMCV is now classified in Pistolviridae. All these viruses infecting multicellular hosts encode proteins that are believed to be involved in extracellular transmission. In PMCV, this relates to a protein of size 33.4 kDa (p33) encoded by a unique third open reading frame. To investigate its characteristics and role, we expressed various recombinant variants of p33 in cultured cells. Our results demonstrate that p33 expression induces a cytotoxic phenotype in transfected cells. The full-length protein undergoes processing into smaller peptide variants. Previous in silico analysis predicted an N-terminal chemokine-like domain, and our present results show that this domain is secreted as peptides capable of inducing cytotoxicity when expressed alone. The C-terminal region includes sequence characteristics of a small hydrophobic domain, which appears crucial for the correct processing of the full-length protein into N- and C-terminal peptides and directing the C-terminal peptides to a high membrane concentration. Investigations into p33 function could elucidate how PMCV achieves extracellular transmission, a mechanism that may be conserved among viruses of Pistolviridae. The findings in this study provide evidence that p33 has structural and functional characteristics of a protein adapted to facilitate host cell membrane interaction and cell lysis, potentially enabling extracellular viral release. These insights may provide evolutionary evidence that pistolviruses have acquired the uncommon trait of viral transmission within the order Ghabrivirales, broadening our understanding of virus–host adaptation in vertebrates.

• Kärkkäinen, Virve; Saari, Toni; Rusanen, Minna; Uusitalo, Hannu; Leinonen, Ville & Thiede, Bernd [Vis alle 9 forfattere av denne artikkelen] (2025). Neuroinflammation Markers in Tear Fluid of Mild Alzheimer’s Disease. Journal of Molecular Neuroscience. ISSN 0895-8696. 75(2). doi: 10.1007/s12031-025-02368-x. Fulltekst i vitenarkiv Vis sammendrag

  The protein composition of tear fluid (TF) reflects the severity and progression of many age-related diseases. Here, we evaluated TF proteins from patients with mild Alzheimer’s disease (AD) and cognitively healthy controls (CO) to explore potential new biomarker molecules. The aim of this study was to explore potential new biomarker molecules by examining the expression of TF proteins whose function is related to neuroinflammation. We examined 53 participants (34 COs, mean age 71 years, Mini-Mental State Examination (MMSE) score 28.9 ± 1.4; 19 with AD, Clinical Dementia Rating 0.5–1, mean age 72 years, MMSE 23.8 ± 2.8). All participants underwent neurological status examination, cognitive testing, and ophthalmological examination. TF was collected using Schirmer strips, and TF protein content was evaluated using mass spectrometry-based proteomics and label-free quantification. We report 14 TF proteins that showed altered protein expression in the AD group compared to the CO group. Twelve proteins were significantly upregulated (SERPINA3, FGA, SIAS, ORM1, ANXA3, G6PI/NLK, CH3L2, MSLN, CPPED1, JCHAIN, IGHV5-51, SPARCL1) and two were downregulated (PIP, SCGB2A1) (p ≤ 0.05). Observed altered expression of TF proteins in the AD group may have potential in AD pathology. Since inflammation is one of the earliest signs of neurodegeneration in AD, these proteins are putative new biomarker candidates of early AD.

• Roman, Giacomo; Lauritzen, Knut Husø; Harrison, Sean Philip; Bhattacharya, Anindita; Andresen, Marianne Seierstad & Mowinckel, Marie-Christine [Vis alle 16 forfattere av denne artikkelen] (2025). Ex vivo correction of severe coagulation Factor VII deficiency in patient-derived 3D liver organoids. Haematologica. ISSN 0390-6078. doi: 10.3324/haematol.2025.288046. Fulltekst i vitenarkiv Vis sammendrag

  Coagulation factor (F) VII deficiency is the most frequent among the rare, inherited bleeding disorders and is predominantly caused by missense mutations in the F7 gene. The disease phenotype ranges from asymptomatic cases to extremely severe hemorrhagic forms, requiring prophylactic injections with plasma-derived or recombinant FVII concentrates. In response, we have developed an autologous cell-based approach that corrects the disease-causing mutation in patient-derived induced pluripotent stem cells (iPSCs) and generates therapeutic, three-dimensional hepatic organoids (HOs). We report the CRISPRmediated correction of homozygous c.718G>C (p.G240R), a missense mutation associated with a severe, life-threatening bleeding phenotype. The HOs contain all liver cell types and exhibit key liver functions, including coagulation factor production. After correction, our data indicate that the patient-derived HOs secrete consistent amounts of functional FVII protein, resulting in improved thrombin generation times. These results represent a significant milestone toward the establishment of an autologous cell-based therapy for patients with FVII- and other coagulation factor deficiencies.

• Haugsten, Helene Rygvold; Kristoffersen, Anne Karin; Enersen, Morten; Thiede, Bernd; Søland, Tine Merete & Haug, Trude M. [Vis alle 7 forfattere av denne artikkelen] (2025). Bacterial extracellular vesicles from Porphyromonas gingivalis – proteomic profile and influence on oral fibroblasts. Archives of Oral Biology. ISSN 0003-9969. 181. doi: 10.1016/j.archoralbio.2025.106424. Fulltekst i vitenarkiv Vis sammendrag

  Objectives In this study, we aimed to explore the protein content of bacterial extracellular vesicles (bEVs) from three strains of Porphyromonas gingivalis, an anaerobic gram-negative bacterium closely associated with periodontitis. Additionally, we aimed to investigate the effects of bEV uptake on human oral fibroblasts, to better understand the role of these vesicles in the pathogenesis of periodontitis. Design Liquid chromatography-mass spectrometry was performed on bEVs from three strains of P. gingivalis with different virulence: ATCC 33277, A7A1–28, and W83. Three key proteins associated with virulence were validated using dot blot. Proliferation and viability of human oral fibroblasts after incubation with bEVs were assessed at different timepoints and different concentrations of bEVs. Results A total of 307 proteins were identified in the bEVs from the three strains of P. gingivalis. Among those were several relevant for virulence, for example gingipains, fimbriae, and peptidyl-arginine deiminase (PPAD). When oral fibroblasts were exposed to bEVs from A7A1–28 or W83, proliferation was significantly affected after 12 and 24 h at one bEV concentration. Furthermore, viability was significantly affected by bEVs from W83 at several timepoints and bEV concentrations. bEVs from ATCC 33277 did not induce any significant changes. Conclusion Several proteins associated with virulence were detected in bEVs from all three strains of P. gingivalis, with varying abundance. Uptake of these bEVs can influence human cells, as exemplified by changes in viability and proliferation.

• Kärkkäinen, Virve; Saari, Toni; Hannonen, Sanna; Rusanen, Minna; Lehtola, Juha Matti & Uusitalo, Hannu [Vis alle 11 forfattere av denne artikkelen] (2025). Altered tear fluid protein expression in persons with mild Alzheimer's disease in proteins involved in oxidative stress, protein synthesis, and energy metabolism. Journal of Alzheimer's Disease. ISSN 1387-2877. 105(1), s. 292–301. doi: 10.1177/13872877251326868. Fulltekst i vitenarkiv Vis sammendrag

  Background Tear fluid (TF) is a protein-rich solution that reflects pathophysiological changes in Alzheimer's disease (AD). Objective In this study, we examined whether TF proteins were differently expressed in persons with mild AD dementia compared to cognitively healthy controls (CO). Methods We analyzed data from 53 study participants including 34 CO (mean age, 71 years; Mini-Mental State Examination [MMSE] score, 28.9 ± 1.4), and 19 patients with AD (Clinical Dementia Rating, 0.5–1; mean age, 72 years; MMSE score, 23.8 ± 2.8). All participants underwent cognitive testing, as well as neurological and ophthalmological examinations. TF was collected using Schirmer strips, and TF protein content was evaluated using mass spectrometry-based proteomics and label-free quantification. Results We found that 16 proteins exhibited significantly upregulated expression in the AD group compared to the CO group (p ≤ 0.05). These proteins were NP1L4, BBOX1, CYTC, RNAS4, PCD, RNT2, AL1A3, SYSC, TPIS, CLH1, PGAM1, EIF3L, 5NTC, HNRNPA2B1, PYGL, and ERO1α. No proteins were significantly downregulated in the AD group compared to the CO group. Conclusions Our results support the hypothesis that TF is a potential source of biomarkers for AD. Part of those proteins with altered expression have previously linked to increased oxidative stress, changed protein synthesis, and disturbed regulation of energy metabolism related to AD or neurodegenerative disease. The present results indicate the value of continued investigation of TF proteins in AD.

• Lauretis, Angela de; Wang, Qiang; Santacroce, Marco; Thiede, Bernd; Ma, Qianli & Lyngstadaas, Ståle Petter [Vis alle 9 forfattere av denne artikkelen] (2025). A Comparative Study of the Impact of Chemical Debridement Products on the Proteomic Profile of the Salivary Pellicle and Cell Adhesion on OsseoSpeed Titanium Dental Implant Surfaces. ACS Applied Materials & Interfaces. ISSN 1944-8244. 17, s. 54579–54592. doi: 10.1021/acsami.5c14570. Fulltekst i vitenarkiv Vis sammendrag

  Background and objectives: Dental implants are commonly used for tooth replacement, with excellent success rates over 10 years. However, exposure to oral cavity bacteria makes them susceptible to biofilm accumulation, which can lead to peri-implant diseases. The salivary pellicle, a protein-rich layer formed on implant surfaces, mediates bacterial adhesion and cell attachment. Effective decontamination is essential for managing bacterial infections, yet no consensus exists on the best chemical agent. Understanding how chemical agents affect the pellicle proteomic profile may guide the selection of optimal treatments. Methods: We investigate the impact of chemical decontamination agents on the salivary pellicle on OsseoSpeed-like titanium dental implant surfaces using a BCA assay and proteomics, and on cell adhesion using human gingival fibroblasts (HGF) and human bone marrow mesenchymal stem cells (hBMSC). We compare the effects of hydrogen peroxide (H2O2), Poloxamer 407 (P407), P407 + H2O2 and sodium hypochlorite + amino acids (NaOCl + AA) using the salivary pellicle and a clean titanium surface as controls. By quantifying and identifying proteins on titanium surfaces after decontamination, this study reveals how chemical agents affect the salivary pellicle. Results: All products reduced the total protein on the surfaces, with the P407 + H2O2 hydrogel demonstrating lower protein variety and superior effectiveness in preventing surface recontamination. Not all proteins were removed during decontamination procedures, and unique proteins were detected in each experimental condition, suggesting protein readsorption depends on surface chemistry. P407 + H2O2 shows the highest enrichment in integrin- and cadherin-binding proteins upon recontamination. P407 and P407 + H2O2 promoted HGFs and hBMSCs attachment. Conversely, NaOCl + AA and H2O2 showed lower HGFs and hBMSCs counts. Conclusion: The P407 + H2O2 hydrogel had the strongest decontamination effect and limited surface recontamination. Each chemical agent produced a distinct proteomic profile. P407 and P407 + H2O2 promoted initial cell adhesion.

• Golnarnik, Golnaz; Thiede, Bernd; Søland, Tine Merete; Galtung, Hilde & Haug, Trude Marie (2025). Hydrogen peroxide-induced oxidative stress alters protein expression in two rat salivary acinar cell lines. Archives of Oral Biology. ISSN 0003-9969. 175. doi: 10.1016/j.archoralbio.2025.106254. Fulltekst i vitenarkiv Vis sammendrag

  Objectives: This study aimed to investigate the impact of hydrogen peroxide-induced oxidative stress on the protein expression profiles of submandibular and parotid acinar cells using a proteomic approach. We sought to evaluate how oxidative stress might contribute to salivary gland dysfunction and whether the two glands respond differently.<p> <p>Design: Immortalized rat parotid gland (PG) and submandibular gland (SMG) acinar epithelial cell lines were exposed to 50 µM and 150 µM hydrogen peroxide for 24 hr, followed by protein identification and quantification via liquid chromatography-mass spectrometry. Immunofluorescence microscopy and western blot analysis validated selected protein expressions, and cell viability was assessed using trypan blue exclusion assays. <p>Results: Compared to controls, histone H4 expression increased in both cell types after hydrogen peroxide exposure, whereas voltage-dependent anion-selective channel 1, keratin 7, and keratin 8 increased only in parotid gland cells. Conversely, mitochondrial aldehyde dehydrogenase and kidney isoform glutaminase were downregulated in parotid gland cells. Basal expression of mitochondrial aldehyde dehydrogenase and catalase was higher in submandibular gland cells. At higher hydrogen peroxide concentrations, antioxidant proteins expression and cell viability were greater in submandibular gland cells compared to parotid gland cells. <p>Conclusions: Our results suggest that submandibular gland acinar cells exhibit greater resistance to oxidative stress compared to parotid gland cells, potentially due to distinct antioxidant and metabolic coping strategies. Understanding these gland-specific responses may contribute to future approaches to protect salivary glands from oxidative damage under pathological conditions.

• Ma, Qianli; Piaia, Lya; Loča, Dagnija; Rubenis, Kristaps; Locs, Janis & Thiede, Bernd [Vis alle 8 forfattere av denne artikkelen] (2024). Soluble Proteins From Conventional and Organic Eggshell Membranes With Different Proteomic Profiles Show Similar In Vitro Biofunctions. Journal of Biomedical Materials Research. Part A. ISSN 1549-3296. 113(1). doi: 10.1002/jbm.a.37848. Fulltekst i vitenarkiv Vis sammendrag

  The eggshell membrane (ESM), resembling the extracellular matrix, acts as a protective barrier against bacterial invasion and offers various biofunctions due to its porous structure and protein-rich composition, such as ovalbumin, ovotransferrin, collagen, soluble protein, and antimicrobial proteins. However, ESM's structure primarily comprises disulfide bonds and heterochains, challenging protein solubilization/extraction. Therefore, the method of dissolving and extracting bioactive protein components in ESM has extremely high potential value and importance for exploring the reuse of egg waste and environmental protection. In this study we extracted soluble ESM proteins (SEPs) from conventional (industrial-fed) and organic (free-grounded) using an acidic 3-mercaptopropionic acid (3-MPA) extraction strategy. FTIR was used to monitor the chemical changes in the ESM, and LC-MS/MS was employed to conduct the proteomic analysis. The biocompatibility and the effects of SEPs cocktails on extracellular matrix (ECM) synthesis were also investigated. The results indicated that the acidic 3-MPA strategy could effectively alter the ESM chemical composition, thereby facilitating SEPs extraction. The SEPs from conventional and organic eggs have different protein profiles but with partial overlapping. SEPs from different sources exhibited similarly desirable biosafety profiles and similar dose-dependent promotion of osteoblastic (ECM) component synthesis, suggesting that different egg sources may contribute to consistent core biological functions of protein products but may also introduce different functional priorities

• Mrsa, Ago; Nardini, Greta; Halvorsen, Trine Grønhaug; Thiede, Bernd & Reubsaet, Leon (2024). One-step functionalization of paper and simplified antibody immobilization for on-the-spot immunocapture from dried serum in liquid chromatography-tandem mass spectrometry based targeted protein determination. Journal of Mass Spectrometry. ISSN 1076-5174. 59(1). doi: 10.1002/jms.4989. Fulltekst i vitenarkiv Vis sammendrag

  This work aimed to simplify and improve the process of binding monoclonal antibodies (mAbs) covalently to filter paper for use in dried blood spot sampling, enabling instant capture of protein biomarkers for targeted protein determination. Incorporating the necessary immunocapture sample preparation step in the initial sampling stage saves time and reduces the workload. The biomarker human chorionic gonadotropin (hCG) was used as the model analyte. The antibody-based paper samplers were prepared by functionalizing paper discs (6 mm) through a simple reaction using divinyl sulfone (DVS). After DVS activation, the paper discs were incubated with E27 hCG mAbs, followed by 0.05% tween/phosphate buffer saline to block the surface. After sample application and drying, the discs only needed to be washed before tryptic digestion and finally analysed on a nanoliquid chromatography–tandem mass spectrometry system. The finished DVS-mAbs samplers could selectively capture hCG (100 ng/mL) from human serum, with a recovery of 50%. Sample clean-up reduced the number of identified proteins from 132 to 82 before and after wash, respectively, with a 70% reduction in serum albumin signal while still retaining hCG on the sampler during the washing protocol. An evaluation of the samplers revealed excellent linearity (R2 = 0.9995) for hCG in serum with relative standard deviations below 15%. This work has presented the first ever reported paper samplers immobilized with antibodies utilizing DVS chemistry, showing promise in the future of paper-based sampling.

• Johansen, Anette; Thiede, Bernd; Anonsen, Jan Haug & Nilsson, Göran Erik (2024). Phosphoproteomic changes in response to anoxia are tissue-specific in the anoxia-tolerant crucian carp (Carassius carassius). Frontiers in Physiology. 15. doi: 10.3389/fphys.2024.1407834. Fulltekst i vitenarkiv Vis sammendrag

  Crucian carp (Carassius carassius), a freshwater fish, can survive chronic anoxia for several months at low temperatures. Consequently, anoxia-related physiological and biochemical adaptations in this species have been studied for more than half a century. Still, despite for the well-known role of protein phosphorylation in regulating cellular processes, no studies have comprehensively characterized the phosphoproteome in crucian carp. In this study, we report the global phosphoproteome in crucian carp brain and liver during anoxia and reoxygenation. By applying a bottom-up proteomic approach on enriched phosphopeptides we found that the brain phosphoproteome shows surprisingly few changes during anoxia-reoxygenation exposure with only 109 out of 4200 phosphopeptides being differentially changed compared to normoxic controls. By contrast, in the liver 395 out of 1287 phosphopeptides changed. Although most changes occurred in the liver phosphoproteome, the pattern of changes indicated metabolic depression and decreased translation in both brain and liver. We also found changes in phosphoproteins involved in apoptotic regulation and reactive oxygen species handling in both tissues. In the brain, some of the most changed phosphopeptides belonged to proteins involved in central nervous system development and neuronal activity at the synaptic cleft. Changed phosphoproteins specific for liver tissue were related to glucose metabolism, such as glycolytic flux and glycogenolysis. In conclusion, protein phosphorylation in response to anoxia and reoxygenation showed both common and tissue-specific changes related to the functional differences between brain and liver.

• Bjørnestad, Synne Arstad; Solbakken, Monica Hongrø; Krokene, Pia; Thiede, Bernd; Hylland, Ketil & Jakobsen, Kjetill Sigurd [Vis alle 9 forfattere av denne artikkelen] (2024). The Atlantic Cod MHC I compartment has the properties needed for cross-presentation in the absence of MHC II. Scientific Reports. 14(1). doi: 10.1038/s41598-024-76225-z. Fulltekst i vitenarkiv Vis sammendrag

  Atlantic cod has a peculiar immune system, characterized by the loss of Major Histocompatibility Complex (MHC) class II pathway, and an extreme expansion of the MHC class I gene repertoire. This has led to the hypothesis that some of the MHC I variants have replaced MHC II by presenting exogenous-peptides in a process similar to cross-presentation. In mammals, MHC I loads endogenous antigens in the endoplasmic reticulum, but we recently found that different Atlantic cod MHC I gene variants traffic to endolysosomes. There, they colocalize with Tapasin and other components of the peptide-loading complex, indicating a plausible peptide-loading system outside the endoplasmic reticulum. In this study, we further characterize the identity of the Atlantic cod MHC I compartment (cMIC). We found that, similarly to mammalian MHC II compartment, cMIC contains late endosomal markers such as Rab7, LAMP1 and CD63. Furthermore, we identified Hsp90b1 (also known as grp94) and LRP1 (also known as CD91) as interactors of MHC I by mass spectrometry. As these two proteins are involved in cross-presentation in mammals, this further suggests that Atlantic cod MHC I might use a similar mechanism to present exogenous peptides, thus, compensating for the absence of MHC II.

• Winiarczyk, Mateusz; Thiede, Bernd; Utheim, Tor Paaske; Kaarniranta, Kai; Winiarczyk, Dagmara & Michalak, Katarzyna [Vis alle 7 forfattere av denne artikkelen] (2024). Oxidative Stress, Persistent Inflammation and Blood Coagulation Alterations in Serum Proteome of Patients with Neovascular Age-Related Macular Degeneration. Life. 14(5). doi: 10.3390/life14050624. Fulltekst i vitenarkiv Vis sammendrag

  Neovascular age-related macular degeneration (AMD) is a major cause of irreversible blindness in elderly populations in developed countries. AMD’s etiopathology is multifactorial, with strong environmental and genetic components, but the exact molecular pathomechanisms underlying the disease are still unknown. In this study, we analyzed blood serum collected from 74 neovascular AMD patients and 58 healthy controls to identify proteins that may serve as potential biomarkers and expand our knowledge about the etiopathogenesis of the disease. The study revealed 17 differentially expressed proteins—11 up-regulated and 6 down-regulated—in neovascular AMD, which are involved in the biological processes previously linked with the disease—oxidative stress and persistent inflammation, impaired cellular transport, lipid metabolism and blood coagulation. In conclusion, the differences in the expressions of the proteins identified in this study may contribute to our understanding of the mechanisms underlying AMD and possibly serve in future as promising biomarkers.

• Kärkkäinen, Virve; Hannonen, Sanna; Rusanen, Minna; Lehtola, Juha-Matti; Saari, Toni & Uusitalo, Hannu [Vis alle 11 forfattere av denne artikkelen] (2024). Tear fluid reflects the altered protein expressions of Alzheimer's disease patients in proteins involved in protein repair and clearance system or the regulation of cytoskeleton. Journal of Alzheimer's Disease. ISSN 1387-2877. doi: 10.1177/13872877241295315. Fulltekst i vitenarkiv
• Storås, Andrea; Magnø, Morten Schjerven; Fineide, Fredrik; Thiede, Bernd; Chen, Xiangjun & Strumke, Inga [Vis alle 9 forfattere av denne artikkelen] (2023). Identifying Important Proteins in Meibomian Gland Dysfunction with Explainable Artificial Intelligence. IEEE International Symposium on Computer-Based Medical Systems. doi: 10.1109/CBMS58004.2023.00217. Fulltekst i vitenarkiv Vis sammendrag

  Meibomian gland dysfunction is the most common cause of dry eye disease and leads to significantly reduced quality of life and social burdens. Because meibomian gland dysfunction results in impaired function of the tear film lipid layer, studying the expression of tear proteins might increase the understanding of the etiology of the condition. Machine learning is able to detect patterns in complex data. This study applied machine learning to classify levels of meibomian gland dysfunction from tear proteins. The aim was to investigate proteomic changes between groups with different severity levels of meibomian gland dysfunction, as opposed to only separating patients with and without this condition. An established feature importance method was used to identify the most important proteins for the resulting models. Moreover, a new method that can take the uncertainty of the models into account when creating explanations was proposed. By examining the identified proteins, potential biomarkers for meibomian gland dysfunction were discovered. The overall findings are largely conirmatory, indicating that the presented machine learning approaches are promising for detecting clinically relevant proteins. While this study provides valuable insights into proteomic changes associated with varying severity levels of meibomian gland dysfunction, it should be noted that it was conducted without a healthy control group. Future research could benefit from including such a comparison to further validate and extend the findings presented here.

• Johansen, Anette; Thiede, Bernd; Anonsen, Jan Haug & Nilsson, Göran Erik (2023). Surviving without oxygen involves major tissue specific changes in the proteome of crucian carp (Carassius carassius). PeerJ. 11, s. 1–28. doi: 10.7717/peerj.14890. Fulltekst i vitenarkiv Vis sammendrag

  The crucian carp ( Carassius carassius ) can survive complete oxygen depletion (anoxia) for several months at low temperatures, making it an excellent model for studying molecular adaptations to anoxia. Still, little is known about how its global proteome responds to anoxia and reoxygenation. By applying mass spectrometry-based proteome analyses on brain, heart and liver tissue from crucian carp exposed to normoxia, five days anoxia, and reoxygenation, we found major changes in particularly cardiac and hepatic protein levels in response to anoxia and reoxygenation. These included tissue-specific differences in mitochondrial proteins involved in aerobic respiration and mitochondrial membrane integrity. Enzymes in the electron transport system (ETS) decreased in heart and increased massively in liver during anoxia and reoxygenation but did not change in the brain. Importantly, the data support a special role for the liver in succinate handling upon reoxygenation, as suggested by a drastic increase of components of the ETS and uncoupling protein 2, which could allow for succinate metabolism without excessive formation of reactive oxygen species (ROS). Also during reoxygenation, the levels of proteins involved in the cristae junction organization of the mitochondria changed in the heart, possibly functioning to suppress ROS formation. Furthermore, proteins involved in immune (complement) system activation changed in the anoxic heart compared to normoxic controls. The results emphasize that responses to anoxia are highly tissue-specific and related to organ function.

• Juvkam, Inga Solgård; Zlygosteva, Olga; Sitarz, Mateusz; Thiede, Bernd; Sørensen, Brita Singers & Malinen, Eirik [Vis alle 9 forfattere av denne artikkelen] (2023). Proton Compared to X-Irradiation Induces Different Protein Profiles in Oral Cancer Cells and Their Derived Extracellular Vesicles. International Journal of Molecular Sciences. ISSN 1661-6596. 24(23), s. 1–17. doi: 10.3390/ijms242316983. Fulltekst i vitenarkiv Vis sammendrag

  Extracellular vesicles (EVs) are membrane-bound particles released from cells, and their cargo can alter the function of recipient cells. EVs from X-irradiated cells have been shown to play a likely role in non-targeted effects. However, EVs derived from proton irradiated cells have not yet been studied. We aimed to investigate the proteome of EVs and their cell of origin after proton or X-irradiation. The EVs were derived from a human oral squamous cell carcinoma (OSCC) cell line exposed to 0, 4, or 8 Gy from either protons or X-rays. The EVs and irradiated OSCC cells underwent liquid chromatography–mass spectrometry for protein identification. Interestingly, we found different protein profiles both in the EVs and in the OSCC cells after proton irradiation compared to X-irradiation. In the EVs, we found that protons cause a downregulation of proteins involved in cell growth and DNA damage response compared to X-rays. In the OSCC cells, proton and X-irradiation induced dissimilar cell death pathways and distinct DNA damage repair systems. These results are of potential importance for understanding how non-targeted effects in normal tissue can be limited and for future implementation of proton therapy in the clinic.

• Storås, Andrea; Fineide, Fredrik; Magnø, Morten Schjerven; Thiede, Bernd; Chen, Xiangjun & Strumke, Inga [Vis alle 11 forfattere av denne artikkelen] (2023). Using machine learning model explanations to identify proteins related to severity of meibomian gland dysfunction. Scientific Reports. 13(1). doi: 10.1038/s41598-023-50342-7. Fulltekst i vitenarkiv Vis sammendrag

  Meibomian gland dysfunction is the most common cause of dry eye disease and leads to significantly reduced quality of life and social burdens. Because meibomian gland dysfunction results in impaired function of the tear film lipid layer, studying the expression of tear proteins might increase the understanding of the etiology of the condition. Machine learning is able to detect patterns in complex data. This study applied machine learning to classify levels of meibomian gland dysfunction from tear proteins. The aim was to investigate proteomic changes between groups with different severity levels of meibomian gland dysfunction, as opposed to only separating patients with and without this condition. An established feature importance method was used to identify the most important proteins for the resulting models. Moreover, a new method that can take the uncertainty of the models into account when creating explanations was proposed. By examining the identified proteins, potential biomarkers for meibomian gland dysfunction were discovered. The overall findings are largely confirmatory, indicating that the presented machine learning approaches are promising for detecting clinically relevant proteins. While this study provides valuable insights into proteomic changes associated with varying severity levels of meibomian gland dysfunction, it should be noted that it was conducted without a healthy control group. Future research could benefit from including such a comparison to further validate and extend the findings presented here.

• Meyer, Saskia; Blaas, Isaac; Bollineni, Ravi Chand; Delic-Sarac, Marina; Tran, Trung & Knetter, Cathrine [Vis alle 20 forfattere av denne artikkelen] (2023). Prevalent and immunodominant CD8 T cell epitopes are conserved in SARS-CoV-2 variants. Cell reports. 42(1). doi: 10.1016/j.celrep.2023.111995. Fulltekst i vitenarkiv Vis sammendrag

  The emergence of SARS-CoV-2 variants of concern (VOC) is driven by mutations that mediate escape from neutralizing antibodies. There is also evidence that mutations can cause loss of T cell epitopes. However, studies on viral escape from T cell immunity have been hampered by uncertain estimates of epitope prevalence. Here, we map and quantify CD8 T cell responses to SARS-CoV-2-specific minimal epitopes in blood drawn from April to June 2020 from 83 COVID-19 convalescents. Among 37 HLA ligands eluted from five prevalent alleles and an additional 86 predicted binders, we identify 29 epitopes with an immunoprevalence ranging from 3% to 100% among individuals expressing the relevant HLA allele. Mutations in VOC are reported in 10.3% of the epitopes, while 20.6% of the non-immunogenic peptides are mutated in VOC. The nine most prevalent epitopes are conserved in VOC. Thus, comprehensive mapping of epitope prevalence does not provide evidence that mutations in VOC are driven by escape of T cell immunity.

• Bogucka-Janczi, Katarzyna; Harms, Gregory; Coissieux, Marie-May; Bentires-Alj, Mohamed; Thiede, Bernd & Rajalingam, Krishnaraj (2023). ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization. eLIFE. 12, s. 1–40. doi: 10.7554/eLife.85167. Fulltekst i vitenarkiv Vis sammendrag

  The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding-proteins and nucleation-promoting factors to perform fundamental cellular functions. We have previously shown that ERK3, an atypical MAPK, controls IL-8 production and chemotaxis (Bogueka et al., 2020). Here, we show in human cells that ERK3 directly acts as a guanine nucleotide exchange factor for CDC42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent RAC1 and CDC42 activation, maintenance of F-actin content, filopodia formation, and epithelial cell migration. Further, ERK3 protein bound directly to the purified ARP2/3 complex and augmented polymerization of actin in vitro. ERK3 kinase activity was required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions.

• Reubsaet, Leon; Thiede, Bernd & Halvorsen, Trine Grønhaug (2022). Next generation VAMS®–Trypsin immobilization for instant proteolysis in bottom-up protein determination. Advances in Sample Preparation. 3. doi: 10.1016/j.sampre.2022.100027. Fulltekst i vitenarkiv Vis sammendrag

  The purpose of this study was to develop a smart proteolysis sampler based on commercially available VAMS® tips employing Volumetric Absorptive Microsampling. In this proof-of-concept paper we show that covalently bound trypsin to VAMS exhibits sufficient activity for proteomic purposes without altering physical conditions. Alkaline pH was needed for the covalent binding of trypsin and at least 50 μg trypsin was required to obtain maximal activity. Prolonged digestion times (up to overnight) of a sample containing 7 model proteins on the VAMS yielded higher peak intensities for signature peptides which are beneficial for quantitative analysis. In-solution digestions of three proteins (carbonic anhydrase 2, catalase and BSA) yielded higher intensities of their signature peptides whereas the other four proteins (cytochrome C, myoglobin, β-lactoglobulin, and phosphorylase B) showed higher intensities for their signature peptides from digestions performed on trypsin modified VAMS. In comparison with conventional in-solution digestions, approximately half of the proteins were identified from diluted serum using trypsin on VAMS. For shotgun serum proteomics, diluted samples collected on trypsin modified VAMS followed by immediate drying yielded similar peptide- (942 vs 875) and protein (96 vs 99) identifications compared to those collected on trypsin modified VAMS followed by overnight digestions. This result shows the potential of trypsin on VAMS digestions.

• Semenova, Daria; Zabirnyk, Arsenii; Lobov, Arseniy; Boyarskaya, Nadezda; Kachanova, Olga & Uspensky, Vladimir [Vis alle 16 forfattere av denne artikkelen] (2022). Multi-omics of in vitro aortic valve calcification. Frontiers in Cardiovascular Medicine. 9. doi: 10.3389/fcvm.2022.1043165. Fulltekst i vitenarkiv Vis sammendrag

  Heart valve calcification is an active cellular and molecular process that partly remains unknown. Osteogenic differentiation of valve interstitial cells (VIC) is a central mechanism in calcific aortic valve disease (CAVD). Studying mechanisms in CAVD progression is clearly needed. In this study, we compared molecular mechanisms of osteogenic differentiation of human VIC isolated from healthy donors or patients with CAVD by RNA-seq transcriptomics in early timepoint (48 h) and by shotgun proteomics at later timepoint (10th day). Bioinformatic analysis revealed genes and pathways involved in the regulation of VIC osteogenic differentiation. We found a high amount of stage-specific differentially expressed genes and good accordance between transcriptomic and proteomic data. Functional annotation of differentially expressed proteins revealed that osteogenic differentiation of VIC involved many signaling cascades such as: PI3K-Akt, MAPK, Ras, TNF signaling pathways. Wnt, FoxO, and HIF-1 signaling pathways were modulated only at the early timepoint and thus probably involved in the commitment of VIC to osteogenic differentiation. We also observed a significant shift of some metabolic pathways in the early stage of VIC osteogenic differentiation. Lentiviral overexpression of one of the most upregulated genes (ZBTB16, PLZF) increased calcification of VIC after osteogenic stimulation. Analysis with qPCR and shotgun proteomics suggested a proosteogenic role of ZBTB16 in the early stages of osteogenic differentiation.

• Cui, Xueyan; Stavik, Benedicte; Thiede, Bernd; Sandset, Per Morten & Kanse, Sandip (2022). FSAP Protects against Histone-Mediated Increase in Endothelial Permeability In Vitro. International Journal of Molecular Sciences. ISSN 1661-6596. 23(22). doi: 10.3390/ijms232213706. Fulltekst i vitenarkiv Vis sammendrag

  Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, we have investigated the effects of FSAP on endothelial permeability in vitro. A mixture of histones from calf thymus stimulated permeability, and the wild-type (WT) serine protease domain (SPD) of FSAP blocked this effect. WT–SPD–FSAP did not influence permeability on its own, nor that stimulated by thrombin or vascular endothelial growth factor (VEGF)-A165. Histones induced a large-scale rearrangement of the junction proteins VE-cadherin and zona occludens-1 from a clear junctional distribution to a diffuse pattern. The presence of WT–SPD–FSAP inhibited these changes. Permeability changes by histones were blocked by both TLR-2 and TLR4 blocking antibodies. Histones upregulated the expression of TLR-2, but not TLR-4, in HUVEC cells, and WT–SPD–FSAP abolished the upregulation of TLR-2 expression. An inactive variant, Marburg I (MI)–SPD–FSAP, did not have any of these effects. The inhibition of histone-mediated permeability may be an important function of FSAP with relevance to sepsis, trauma, and stroke and the need to be investigated further in in vivo experiments.

• Nguyen, Minh Thao; Halvorsen, Trine Grønhaug; Thiede, Bernd & Reubsaet, Leon (2022). Smart proteolysis samplers for pre-lab bottom-up protein analysis – Performance of on-paper digestion compared to conventional digestion. Separation Science Plus (SSC plus). 5(5), s. 171–183. doi: 10.1002/sscp.202100062. Fulltekst i vitenarkiv Vis sammendrag

  Here the relation between digestion of proteins by trypsin covalently bound to paper and trypsin in-solution is investigated. The trypsin acting on paper is covalently bound. A trypsin concentration of 0.5% (w/v) results in the highest digestion activity of all concentrations tested. Additionally, it can be seen that trypsin on-paper has retained approx. 50% of its activity. Unlike trypsin in-solution, the stability of the smart proteolysis samplers was regarded to be stable for at least four months when kept refrigerated. Autolysis was very small for covalently bound trypsin: less than 2% compared to in-solution trypsin. Proteomic analysis of diluted human serum showed more protein identifications (214) in-solution digestions than on-paper digestions (76). Also, higher coverage for the in-solution digestion was obtained. Those proteins identified after on-paper digestion with no or few disulfide bonds seem to have more similar sequence coverages compared to those identified after in-solution digestion. Smart samplers allow the determination of at least 70–75 proteins without performing the overnight digestion. All in all, trypsin covalently bound to paper shows to retain high proteolytic activity and is a stable alternative for conventional digestions. In this way, smart proteolytic samplers show their feasibility in pre-lab sample preparation.

• Berge-Seidl, Sebastian; Nielsen, Nis Valentin; Alfonso, Armando A. Rodriguez; Etscheid, Michael; Kandanur, Sai Priya Sarma & Haug, Bengt Erik [Vis alle 15 forfattere av denne artikkelen] (2022). Identification of a phage display-derived peptide interacting with the N-terminal region of factor VII activating protease (FSAP) enables characterization of zymogen activation. ACS Chemical Biology. ISSN 1554-8929. 17(9), s. 2631–2642. doi: 10.1021/acschembio.2c00538. Fulltekst i vitenarkiv Vis sammendrag

  Factor VII Activating protease (FSAP) has a protective effect in diverse disease conditions as inferred from studies in FSAP–/– mice and humans deficient in FSAP activity due to single-nucleotide polymorphism. The zymogen form of FSAP in plasma is activated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear whether this activation mechanism is specific and amenable to manipulation. Using a phage display approach, we have identified a Cys-constrained 11 amino acid peptide, NNKC9/41, that activates pro-FSAP in plasma. The synthetic linear peptide has a propensity to cyclize through the terminal Cys groups, of which the antiparallel cyclic dimer, but not the monocyclic peptide, is the active component. Other commonly found zymogens in the plasma, related to the hemostasis system, were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30–35) in an intrinsically disordered stretch in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41, and this was reversed by MA-FSAP-38C7, demonstrating the utility of this peptide. Peptide NNKC9/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP, elucidate its biological role, and provide a starting point for the pharmacological manipulation of FSAP activity.

• Berg, Henriette Engen; Halldórsso, Skarphéðinn; Bakketeig, Eris Aas; Thiede, Bernd; Sandberg, Cecilie & Lundanes, Elsa [Vis alle 8 forfattere av denne artikkelen] (2022). Micro‐pillar array columns (µPAC): An efficient tool for comparing tissue and cultured cells of glioblastoma. Journal of chromatography open. ISSN 2772-3917. 2. doi: 10.1016/j.jcoa.2022.100047. Fulltekst i vitenarkiv
• Hynne, Håvard; Aqrawi, Lara A.; Jensen, Janicke Liaaen; Thiede, Bernd; Palm, Øyvind & Amdal, Cecilie Delphin [Vis alle 10 forfattere av denne artikkelen] (2022). Proteomic Profiling of Saliva and Tears in Radiated Head and Neck Cancer Patients as Compared to Primary Sjögren’s Syndrome Patients. International Journal of Molecular Sciences. ISSN 1661-6596. 23(7). doi: 10.3390/ijms23073714. Fulltekst i vitenarkiv Vis sammendrag

  Patients with head and neck cancer (HNC) and patients with primary Sjögren’s syndrome (pSS) may exhibit similar symptoms of dry mouth and dry eyes, as a result of radiotherapy (RT) or a consequence of disease progression. To identify the proteins that may serve as promising disease biomarkers, we analysed saliva and tears from 29 radiated HNC patients and 21 healthy controls, and saliva from 14 pSS patients by mass spectrometry-based proteomics. The study revealed several upregulated, and in some instances overlapping, proteins in the two patient groups. Histone H1.4 and neutrophil collagenase were upregulated in whole saliva of both patient groups, while caspase-14, histone H4, and protein S100-A9 were upregulated in HNC saliva only. In HCN tear fluid, the most highly upregulated protein was mucin-like protein 1. These overexpressed proteins in saliva and tears play central roles in inflammation, host cell injury, activation of reactive oxygen species, and tissue repair. In conclusion, the similarities and differences in overexpressed proteins detected in saliva from HNC and pSS patients may contribute to the overall understanding of the different pathophysiological mechanisms inducing dry mouth. Thus, the recurring proteins identified could possibly serve as future promising biomarkers

• Kogler, Stian; Aizenshtadt, Aleksandra; Harrison, Sean; Skottvoll, Frøydis Sved; Berg, Henriette Engen & Abadpour, Shadab [Vis alle 14 forfattere av denne artikkelen] (2022). "Organ-in-a-Column" Coupled On-line with Liquid Chromatography-Mass Spectrometry. Analytical Chemistry. ISSN 0003-2700. 94(50), s. 17677–17684. doi: 10.1021/acs.analchem.2c04530. Fulltekst i vitenarkiv Vis sammendrag

  Organoids, i.e., laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling, and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the “organ-in-a-column” units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were coloaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated “on column” with heroin, followed by on-line monitoring of the drug’s phase 1 metabolism. Enzymatic metabolism of heroin produced in the “organ-in-a-column” units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.

• Kallas, Pawel; Valen, Håkon; Hulander, Mats; Gadegaard, Nikolaj; Stormonth-Darling, John & O'Reilly, Padraic [Vis alle 9 forfattere av denne artikkelen] (2022). Protein-coated nanostructured surfaces affect the adhesion of Escherichia coli. Nanoscale. ISSN 2040-3364. 14(20), s. 7736–7746. doi: 10.1039/d2nr00976e. Fulltekst i vitenarkiv Vis sammendrag

  Developing new implant surfaces with anti-adhesion bacterial properties used for medical devices remains a challenge. Here we describe a novel study investigating nanotopography influences on bacterial adhesion on surfaces with controlled interspatial nanopillars distances. Surfaces were coated with proteins (fibrinogen, collagen, serum and saliva) prior to E. coli-WT adhesion under flow conditions. PiFM provided chemical mapping and showed that proteins adsorbed both between and onto the nanopillars with a preference to areas between the nanopillars. E. coli-WT adhered least to protein-coated areas with low surface nanopillar coverage, most to surfaces coated with saliva, while human serum led to the lowest adhesion. Protein-coated nanostructured surfaces affected the adhesion of E. coli-WT. Abstract text goes here.

• Johannsen, Christina; Koehler, Christian & Thiede, Bernd (2021). Comparison of LFQ and IPTL for Protein Identification and Relative Quantification. Molecules. ISSN 1431-5157. 26(17). doi: 10.3390/molecules26175330. Fulltekst i vitenarkiv Vis sammendrag

  (1) Background: Mass spectrometry-based quantitative proteome profiling is most commonly performed by label-free quantification (LFQ), stable isotopic labeling with amino acids in cell culture (SILAC), and reporter ion-based isobaric labeling methods (TMT and iTRAQ). Isobaric peptide termini labeling (IPTL) was described as an alternative to these methods and is based on crosswise labeling of both peptide termini and MS2 quantification. High quantification accuracy was assumed for IPTL because multiple quantification points are obtained per identified MS2 spectrum. A direct comparison of IPTL with other quantification methods has not been performed yet because IPTL commonly requires digestion with endoproteinase Lys-C. (2) Methods: To enable tryptic digestion of IPTL samples, a novel labeling for IPTL was developed that combines metabolic labeling (Arg-0/Lys-0 and Arg-d4/Lys-d4, respectively) with crosswise N-terminal dimethylation (d4 and d0, respectively). (3) Results: The comparison of IPTL with LFQ revealed significantly more protein identifications for LFQ above homology ion scores but not above identity ion scores. (4) Conclusions: The quantification accuracy was superior for LFQ despite the many quantification points obtained with IPTL.

• Thiede, Bernd & Koehler, Christian (2021). An Approach for Triplex-IPTL. I Marcus, Katrin; Eisenacher, Martin & Sitek, Barbara (Red.), Quantitative Methods in Proteomics. Humana Press. ISSN 9781071610237. s. 133–144. doi: 10.1007/978-1-0716-1024-4_10. Fulltekst i vitenarkiv Vis sammendrag

  Isobaric peptide termini labeling (IPTL) is an approach for quantitative proteomics based on crosswise isotopic labeling of peptides at the N- and C-terminus. The labeling reagents are chosen in isotopic variations that the resulting mass of all labels per peptide is isobaric, but the individual label on each peptide terminus is different. Therefore, the quantitative difference of the peptide signal can be determined by the fragment ions of the corresponding MS2 spectra. Here, we describe an approach for triplex-IPTL to allow the comparison of three proteomes. This approach is based on digestion of the proteins by endoproteinase Lys-C, followed by three combinations of selective dimethylation of the peptide N-termini and subsequent dimethylation of the lysine residues at the C-termini. Data analysis is performed using Mascot for database searches and the freely available software package IsobariQ for quantification.

• Andersen, Elisabeth; Dugarte, Maria Eugenia Chollet; Sletten, Marit; Stavik, Benedicte; Skarpen, Ellen & Backe, Paul Hoff [Vis alle 9 forfattere av denne artikkelen] (2021). Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding. Thrombosis and Haemostasis. ISSN 0340-6245. 121(12), s. 1588–1598. doi: 10.1055/a-1450-8568. Fulltekst i vitenarkiv
• Malecki, Jedrzej Mieczyslaw; O'Donohue, Marie-Francoise; Kim, Yeji; Jakobsson, Magnus; Gessa, Luca & Pinto, Rita [Vis alle 14 forfattere av denne artikkelen] (2021). Human METTL18 is a histidine-specific methyltransferase that targets RPL3 and affects ribosome biogenesis and function. Nucleic Acids Research (NAR). ISSN 0305-1048. 49(6), s. 3185–3203. doi: 10.1093/nar/gkab088. Fulltekst i vitenarkiv Vis sammendrag

  Abstract Protein methylation occurs primarily on lysine and arginine, but also on some other residues, such as histidine. METTL18 is the last uncharacterized member of a group of human methyltransferases (MTases) that mainly exert lysine methylation, and here we set out to elucidate its function. We found METTL18 to be a nuclear protein that contains a functional nuclear localization signal and accumulates in nucleoli. Recombinant METTL18 methylated a single protein in nuclear extracts and in isolated ribosomes from METTL18 knockout (KO) cells, identified as 60S ribosomal protein L3 (RPL3). We also performed an RPL3 interactomics screen and identified METTL18 as the most significantly enriched MTase. We found that His-245 in RPL3 carries a 3-methylhistidine (3MH; τ-methylhistidine) modification, which was absent in METTL18 KO cells. In addition, both recombinant and endogenous METTL18 were found to be automethylated at His-154, thus further corroborating METTL18 as a histidine-specific MTase. Finally, METTL18 KO cells displayed altered pre-rRNA processing, decreased polysome formation and codon-specific changes in mRNA translation, indicating that METTL18-mediated methylation of RPL3 is important for optimal ribosome biogenesis and function. In conclusion, we have here established METTL18 as the second human histidine-specific protein MTase, and demonstrated its functional relevance.

• Guerreiro, Eduarda Mazagão; Øvstebø, Reidun; Thiede, Bernd; Costea, Daniela Elena; Søland, Tine Merete & Galtung, Hilde (2020). Cancer cell line-specific protein profiles in extracellular vesicles identified by proteomics. PLOS ONE. 15(9), s. 1–20. doi: 10.1371/journal.pone.0238591. Fulltekst i vitenarkiv Vis sammendrag

  Extracellular vesicles (EVs), are important for intercellular communication in both physiological and pathological processes. To explore the potential of cancer derived EVs as disease biomarkers for diagnosis, monitoring, and treatment decision, it is necessary to thoroughly characterize their biomolecular content. The aim of the study was to characterize and compare the protein content of EVs derived from three different cancer cell lines in search of a specific molecular signature, with emphasis on proteins related to the carcinogenic process. Oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC) and melanoma brain metastasis cell lines were cultured in CELLine AD1000 flasks. EVs were isolated by ultrafiltration and size-exclusion chromatography and characterized. Next, the isolated EVs underwent liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Functional enrichment analysis was performed for a more general overview of the biological processes involved. More than 600 different proteins were identified in EVs from each particular cell line. Here, 14%, 10%, and 24% of the identified proteins were unique in OSCC, PDAC, and melanoma vesicles, respectively. A specific protein profile was discovered for each cell line, e.g., EGFR in OSCC, Muc5AC in PDAC, and FN1 in melanoma vesicles. Nevertheless, 25% of all the identified proteins were common to all cell lines. Functional enrichment analysis linked the proteins in each data set to biological processes such as “biological adhesion”, “cell motility”, and “cellular component biogenesis”. EV proteomics discovered cancer-specific protein profiles, with proteins involved in processes promoting tumor progression. In addition, the biological processes associated to the melanoma-derived EVs were distinct from the ones linked to the EVs isolated from OSCC and PDAC. The malignancy specific biomolecular cues in EVs may have potential applications as diagnostic biomarkers and in therapy.

• Guldvik, Ingrid Jenny; Zuber, Verena; Braadland, Peder Rustøen; Grytli, Helene Hartvedt; Ramberg, Håkon Andre & Lilleby, Wolfgang [Vis alle 18 forfattere av denne artikkelen] (2020). Identification and Validation of Leucine-rich alpha-2-glycoprotein 1 as a Noninvasive Biomarker for Improved Precision in Prostate Cancer Risk Stratification. European Urology Open Science. ISSN 2666-1691. 21, s. 51–61. doi: 10.1016/j.euros.2020.08.007. Fulltekst i vitenarkiv Vis sammendrag

  Background More accurate risk assessments are needed to improve prostate cancer management. Objective To identify blood-based protein biomarkers that provided prognostic information for risk stratification. Design, setting, and participants Mass spectrometry was used to identify biomarker candidates from blood, and validation studies were performed in four independent cohorts retrospectively collected between 1988 and 2015. Outcome measurements and statistical analysis The primary outcome objectives were progression-free survival, prostate cancer–specific survival (PCSS), and overall survival. Statistical analyses to assess survival and model performance were performed. Results and limitation Serum leucine-rich α-2-glycoprotein 1 (LRG1) was found to be elevated in fatal prostate cancer. LRG1 provided prognostic information independent of metastasis and increased the accuracy in predicting PCSS, particularly in the first 3 yr. A high LRG1 level is associated with an average of two-fold higher risk of disease-progression and mortality in both high-risk and metastatic patients. However, our study design, with a retrospective analysis of samples spanning several decades back, limits the assessment of the clinical utility of LRG1 in today’s clinical practice. Thus, independent prospective studies are needed to establish LRG1 as a clinically useful biomarker for patient management. Conclusions High blood levels of LRG1 are unfavourable in newly diagnosed high-risk and metastatic prostate cancer, and LRG1 increased the accuracy of risk stratification of prostate cancer patients. Patient summary High blood levels of leucine-rich α-2-glycoprotein 1 are unfavourable in newly diagnosed high-risk and metastatic prostate cancer.

• Kara, Emrah; Nielsen, Nis Valentin; Eggertsdottir, Bergrun; Thiede, Bernd; Kanse, Sandip & Løset, Geir Åge (2020). Design and characterization of a new pVII combinatorial phage display peptide library for protease substrate mining using Factor VII activating protease (FSAP) as model. ChemBioChem. ISSN 1439-4227. 21(13), s. 1875–1884. doi: 10.1002/cbic.201900705. Fulltekst i vitenarkiv Vis sammendrag

  We describe a novel, easy and efficient combinatorial phage display peptide substrate‐mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti‐FLAG mAbs and its independency of any post‐translational modification. This library was tested with Factor‐VII activating protease (WT‐FSAP) and its single‐nucleotide polymorphism variant Marburg‐I (MI)‐FSAP. The WT‐FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI‐FSAP as a loss‐of‐function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT‐FSAP substrates than previously anticipated.

• Krishnan, Aswini; Berthelet, Jean; Renaud, Emilie; Rosigkeit, Sebastian; Distler, Ute & Stawiski, Eric [Vis alle 17 forfattere av denne artikkelen] (2020). Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas. Nature Communications. 11. doi: 10.1038/s41467-020-15955-w. Fulltekst i vitenarkiv Vis sammendrag

  Papillary thyroid cancer (PTC) is the most common type of endocrine malignancy. By RNA-seq analysis, we identify a RET rearrangement in the tumour material of a patient who does not harbour any known RAS or BRAF mutations. This new gene fusion involves exons 1–4 from the 5′ end of the Trk fused Gene (TFG) fused to the 3′ end of RET tyrosine kinase leading to a TFG-RET fusion which transforms immortalized human thyroid cells in a kinase-dependent manner. TFG-RET oligomerises in a PB1 domain-dependent manner and oligomerisation of TFG-RET is required for oncogenic transformation. Quantitative proteomic analysis reveals the upregulation of E3 Ubiquitin ligase HUWE1 and DUBs like USP9X and UBP7 in both tumor and metastatic lesions, which is further confirmed in additional patients. Expression of TFG-RET leads to the upregulation of HUWE1 and inhibition of HUWE1 significantly reduces RET-mediated oncogenesis.

• Khan, Ayyad Ahmad Zartasht; Jackson, Catherine; Utheim, Tor Paaske; Reppe, Sjur; Sapkota, Dipak & Olstad, Ole Kristoffer [Vis alle 8 forfattere av denne artikkelen] (2020). Sericin-induced melanogenesis in cultured retinal pigment epithelial cells is associated with elevated levels of hydrogen peroxide and inflammatory proteins. Molecules. ISSN 1431-5157. 25(19), s. 1–20. doi: 10.3390/molecules25194395. Fulltekst i vitenarkiv Vis sammendrag

  We previously demonstrated that the silk protein sericin promotes pigmentation of retinal pigment epithelium (RPE) by activating the NF-κB pathway. Among numerous agents, NF-κB can be activated by hydrogen peroxide. In the present study, we explored possible associations between reactive oxygen species and sericin-induced melanogenesis in RPE. The proteome of human fetal RPE cultured for seven days with or without 1% sericin was analyzed using ingenuity pathway analysis (IPA). The proteomic data was verified by immunofluorescence and immunoblotting. Light microscopy and scanning electron microscopy were used to assess morphology. Dihydroethidium (DHE) and dihydrorhodamine (DHR) assays were used to measure superoxide and hydrogen peroxide species. Expression levels of proteins related to inflammation, differentiation, cell survival and cell adhesion were higher in cells cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 1% sericin, whereas cells cultured in DMEM alone showed higher expression levels of proteins associated with Bruch’s membrane and cytoskeleton. Despite upregulation of inflammatory proteins, sericin co-cultured RPE yielded significantly higher cell viability compared to cells cultured without sericin. Addition of sericin to culture media significantly increased hydrogen peroxide-levels without significantly affecting superoxide-levels. We suggest that sericin-induced melanogenesis in cultured RPE is associated with elevated levels of superoxide dismutase, hydrogen peroxide and inflammatory proteins.

• Solhaug, Anita; Torgersen, Maria Lyngaas; Holme, Jørn Andreas; Wiik-Nielsen, Jannicke; Thiede, Bernd & Eriksen, Gunnar Sundstøl (2020). The Fusarium mycotoxin, 2-Amino-14,16-dimethyloctadecan-3-ol (AOD) induces vacuolization in HepG2 cells. Toxicology. ISSN 0300-483X. s. 433–434. doi: 10.1016/j.tox.2020.152405. Fulltekst i vitenarkiv Vis sammendrag

  The mycotoxin 2-Amino-14,16-dimethyloctadecan-3-ol (AOD) has been isolated from cultures of the fungus Fusarium avenaceum, one of the most prevalent Fusarium species. AOD is an analogue of sphinganine and 1-deoxysphinganine, important intermediates in the de novo biosynthesis of cellular sphingolipids. Here we studied cellular effects of AOD using the human liver cell line HepG2 as a model system. AOD (10 μM) induced a transient accumulation of vacuoles in the cells. The effect was observed at non-cytotoxic concentrations and was not linked to cell death processes. Proteomic analyses indicated that protein degradation and/or vesicular transport may be a target for AOD. Further studies revealed that AOD had only minor effects on the initiation rate of macropinocytosis and autophagy. However, the AOD-induced vacuoles were lysosomal-associated membrane protein-1 (LAMP-1) positive, suggesting that they most likely originate from lysosomes or late endosomes. Accordingly, both endosomal and autophagic protein degradation were inhibited. Further studies revealed that treatment with concanamycin A or chloroquine completely blocked the AOD-induced vacuolization, suggesting that the vacuolization is dependent of acidic lysosomes. Overall, the results strongly suggest that the increased vacuolization is due to an accumulation of AOD in lysosomes or late endosomes thereby disturbing the later stages of the endolysosomal process.

• Perdreau-Dahl, Harmonie; Progida, Cinzia; Barfeld, Stefan Jörg; Guldsten, Hanne; Thiede, Bernd & Arntzen, Magnus Øverlie [Vis alle 10 forfattere av denne artikkelen] (2020). Sjögren syndrome/scleroderma autoantigen 1 is a direct Tankyrase binding partner in cancer cells. Communications Biology. 3(1). doi: 10.1038/s42003-020-0851-2. Fulltekst i vitenarkiv
• Aqrawi, Lara Adnan; Galtung, Hilde; Guerreiro, Eduarda; Øvstebø, Reidun; Thiede, Bernd & Utheim, Tor Paaske [Vis alle 11 forfattere av denne artikkelen] (2019). Proteomic and histopathological characterisation of sicca subjects and primary Sjögren’s syndrome patients reveals promising tear, saliva and extracellular vesicle disease biomarkers. Arthritis Research & Therapy. ISSN 1478-6354. 21, s. 1–14. doi: 10.1186/s13075-019-1961-4. Fulltekst i vitenarkiv Vis sammendrag

  Background Mononuclear cell infiltration of exocrine glands, production of Ro/SSA and La/SSB autoantibodies, along with oral and ocular dryness, are characteristic features of primary Sjögren’s syndrome (pSS). Non-SS sicca subjects, an underexplored group in relation to pSS, display similar sicca symptoms, with possible mild signs of inflammation in their salivary glands, yet with no serological detection of autoantibody production. In this study, we investigated inflammatory manifestations in the salivary gland tissue, tear fluid and saliva of non-SS subjects, as compared to pSS patients and healthy individuals. Methods Fifteen non-SS, 10 pSS and 10 healthy subjects were included in the analyses. Histological evaluation of salivary gland biopsies was performed. Liquid chromatography-mass spectrometry (LC-MS) was conducted on tear fluid and stimulated whole saliva, and proteomic biomarker profiles were generated. Extracellular vesicle (EVs) isolation and characterisation from both fluids were also combined with LC-MS. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold. Database for Annotation, Visualization and Integrated Discovery (DAVID) and Functional Enrichment Analysis Tool (FunRich) were applied for functional analyses. Results Histopathological evaluation of salivary gland biopsies showed implications of milder inflammation in non-SS subjects through mononuclear cell infiltration, fibrosis and fatty replacement, as compared to pSS patients. Although unaffected in the non-SS group, upregulation of proinflammatory pathways and proteins involved in ubiquitination (LMO7 and HUWE1) and B cell differentiation (TPD52) were detected in tear fluid of pSS patients. Moreover, overexpression of proteins STOM, ANXA4 and ANXA1, regulating cellular innate and adaptive immunological pathways, were further identified in EVs from tear fluid of pSS patients. Finally, whole saliva and EVs isolated from whole saliva of pSS patients expressed proteins vital for innate MHC class I cellular regulation (NGAL) and T cell activation (CD44). Conclusions Non-SS sicca subjects may show implications of mild inflammation in their glandular tissue, while their protein profile was strikingly more similar to healthy controls than to pSS patients. Hence, the tear and salivary biomarkers identified could be implemented as potential non-invasive diagnostic tools that may aid in increasing diagnostic accuracy when evaluating non-SS subjects and pSS patients and monitoring disease progression.

• Wagner, Fabienne; Kunz, Tobias C.; Chowdhury, Suvagata R.; Thiede, Bernd; Fraunholz, Martin & Eger, Debora [Vis alle 7 forfattere av denne artikkelen] (2019). Armadillo repeat-containing protein 1 is a dual localization protein associated with mitochondrial intermembrane space bridging complex. PLOS ONE. 14(10), s. 1–19. doi: 10.1371/journal.pone.0218303. Fulltekst i vitenarkiv Vis sammendrag

  Cristae architecture is important for the function of mitochondria, the organelles that play the central role in many cellular processes. The mitochondrial contact site and cristae organizing system (MICOS) together with the sorting and assembly machinery (SAM) forms the mitochondrial intermembrane space bridging complex (MIB), a large protein complex present in mammalian mitochondria that partakes in the formation and maintenance of cristae. We report here a new subunit of the mammalian MICOS/MIB complex, an armadillo repeat-containing protein 1 (ArmC1). ArmC1 localizes both to cytosol and mitochondria, where it associates with the outer mitochondrial membrane through its carboxy-terminus. ArmC1 interacts with other constituents of the MICOS/MIB complex and its amounts are reduced upon MICOS/MIB complex depletion. Mitochondria lacking ArmC1 do not show defects in cristae structure, respiration or protein content, but appear fragmented and with reduced motility. ArmC1 represents therefore a peripheral MICOS/MIB component that appears to play a role in mitochondrial distribution in the cell.

• Riegel, Kristina; Schlöder, Janine; Sobczak, Marco; Jonuleit, Helmut; Thiede, Bernd & Schild, Hansjörg [Vis alle 7 forfattere av denne artikkelen] (2019). RAF kinases are stabilized and required for dendritic cell differentiation and function. Cell Death and Differentiation. ISSN 1350-9047. 27, s. 1300–1315. doi: 10.1038/s41418-019-0416-4. Fulltekst i vitenarkiv Vis sammendrag

  RAF kinases (ARAF, BRAF, and CRAF) are highly conserved enzymes that trigger the RAF-MEK1/2-ERK1/2 (MAPK) pathway upon activation of RAS. Despite enormous clinical interest, relatively little is known on the role of RAFs in mediating immune responses. Here, we investigated the role of RAF kinases and MEK1/2 in dendritic cells (DCs), the central regulators of T cell-mediated antitumor immune responses and the adaptive immune system. We demonstrate that RAF kinases are active and stabilized at their protein levels during DC differentiation. Inhibition of RAF kinases but not MEK1/2 impaired the activation of DCs in both mice and human. As expected, DCs treated with RAF inhibitors show defects in activating T cells. Further, RAF and MEK1/2 kinases are directly required for the activation and proliferation of CD4+ T cells. Our observations suggest that RAF and MEK1/2 have independent roles in regulating DC function that has important implications for administering RAF–MAPK inhibitors in the clinics.

• Itkonen, Harri; Urbanucci, Alfonso; ES, Martin Sara; Khan, Aziz; Mathelier, Anthony & Thiede, Bernd [Vis alle 8 forfattere av denne artikkelen] (2019). High OGT activity is essential for MYC-driven proliferation of prostate cancer cells. Theranostics. 9(8), s. 2183–2197. doi: 10.7150/thno.30834. Fulltekst i vitenarkiv Vis sammendrag

  O-GlcNAc transferase (OGT) is overexpressed in aggressive prostate cancer. OGT modifies intra-cellular proteins via single sugar conjugation (O-GlcNAcylation) to alter their activity. We recently discovered the first fast-acting OGT inhibitor OSMI-2. Here, we probe the stability and function of the chromatin O-GlcNAc and identify transcription factors that coordinate with OGT to promote proliferation of prostate cancer cells. Methods: Chromatin immunoprecipitation (ChIP) coupled to sequencing (seq), formaldehyde-assisted isolation of regulatory elements, RNA-seq and reverse-phase protein arrays (RPPA) were used to study the importance of OGT for chromatin structure and transcription. Mass spectrometry, western blot, RT-qPCR, cell cycle analysis and viability assays were used to establish the role of OGT for MYC-related processes. Prostate cancer patient data profiled for both mRNA and protein levels were used to validate findings. Results: We show for the first time that OGT inhibition leads to a rapid loss of O-GlcNAc chromatin mark. O-GlcNAc ChIP-seq regions overlap with super-enhancers (SE) and MYC binding sites. OGT inhibition leads to down-regulation of SE-dependent genes. We establish the first O-GlcNAc chromatin consensus motif, which we use as a bait for mass spectrometry. By combining the proteomic data from oligonucleotide enrichment with O-GlcNAc and MYC ChIP-mass spectrometry, we identify host cell factor 1 (HCF-1) as an interaction partner of MYC. Inhibition of OGT disrupts this interaction and compromises MYC's ability to confer androgen-independent proliferation to prostate cancer cells. We show that OGT is required for MYC-mediated stabilization of mitotic proteins, including Cyclin B1, and/or the increased translation of their coding transcripts. This implies that increased expression of mRNA is not always required to achieve increased protein expression and confer aggressive phenotype. Indeed, high expression of Cyclin B1 protein has strong predictive value in prostate cancer patients (p=0.000014) while mRNA does not. Conclusions: OGT promotes SE-dependent gene expression. OGT activity is required for the interaction between MYC and HCF-1 and expression of MYC-regulated mitotic proteins. These features render OGT essential for the androgen-independent, MYC-driven proliferation of prostate cancer cells. Androgen-independency is the major mechanism of prostate cancer progression, and our study identifies OGT as an essential mediator in this process.

• Abudu, Yakubu Princely; Pankiv, Serhiy; Mathai, Benan John; Lystad, Alf Håkon; Bindesbøll, Christian & Brenne, Hanne Britt [Vis alle 14 forfattere av denne artikkelen] (2019). NIPSNAP1 and NIPSNAP2 act as “eat-me signal” for mitophagy. Developmental Cell. ISSN 1534-5807. 49(4), s. 509–525.e12. doi: 10.1016/j.devcel.2019.03.013. Fulltekst i vitenarkiv Vis sammendrag

  The clearance of damaged or dysfunctional mitochondria by selective autophagy (mitophagy) is important for cellular homeostasis and prevention of disease. Our understanding of the mitochondrial signals that trigger their recognition and targeting by mitophagy is limited. Here, we show that the mitochondrial matrix proteins 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) and NIPSNAP2 accumulate on the mitochondria surface upon mitochondrial depolarization. There, they recruit proteins involved in selective autophagy, including autophagy receptors and ATG8 proteins, thereby functioning as an “eat me” signal for mitophagy. NIPSNAP1 and NIPSNAP2 have a redundant function in mitophagy and are predominantly expressed in different tissues. Zebrafish lacking a functional Nipsnap1 display reduced mitophagy in the brain and parkinsonian phenotypes, including loss of tyrosine hydroxylase (Th1)-positive dopaminergic (DA) neurons, reduced motor activity, and increased oxidative stress.

• Byskov, Kristina; Gall, Sylvain M. le; Thiede, Bernd; Camerer, Eric & Kanse, Sandip (2019). Protease activated receptors (PAR)‐1 and ‐2 mediate cellular effects of factor VII activating protease (FSAP). The FASEB Journal. ISSN 0892-6638. 34, s. 1079–1090. doi: 10.1096/fj.201801986RR. Fulltekst i vitenarkiv Vis sammendrag

  Factor VII activating protease (FSAP) is a circulating serine protease implicated in thrombosis, atherosclerosis, stroke, and cancer. Using an overexpression strategy, we have systematically investigated the role of protease activated receptors (PAR)‐1, ‐2, ‐3, and ‐4 on FSAP‐mediated signaling in HEK293T and A549 cells. Cleavage of PAR‐reporter constructs and MAPK phosphorylation was used to monitor receptor activation. FSAP cleaved PAR‐2 and to a lesser degree PAR‐1, but not PAR‐3 or PAR‐4 in both cell types. Robust MAPK activation in response to FSAP was observed after PAR‐2, but not PAR‐1 overexpression in HEK293T. Recombinant serine protease domain of wild type FSAP, but not the Marburg I isoform of FSAP, could reproduce the effects of plasma purified FSAP. Canonical cleavage of both PARs was suggested by mass spectrometric analysis of synthetic peptide substrates from the N‐terminus of PARs and site directed mutagenesis studies. Surprisingly, knockdown of endogenous PAR‐1, but not PAR‐2, prevented the apoptosis‐inhibitory effect of FSAP, suggesting that PAR1 is nevertheless a direct or indirect target in some cell types. This molecular characterization of PAR‐1 and ‐2 as cellular receptors of FSAP will help to define the actions of FSAP in the context of cancer and vascular biology.

• Koehler, Christian & Thiede, Bernd (2019). Predominant cleavage of proteins N-terminal to serines and threonines using scandium(III) triflate. JBIC Journal of Biological Inorganic Chemistry. ISSN 0949-8257. 25, s. 61–66. doi: 10.1007/s00775-019-01733-7. Fulltekst i vitenarkiv Vis sammendrag

  Proteolytic digestion prior to LC–MS analysis is a key step for the identification of proteins. Digestion of proteins is typically performed with trypsin, but certain proteins or important protein sequence regions might be missed using this endoproteinase. Only few alternative endoproteinases are available and chemical cleavage of proteins is rarely used. Recently, it has been reported that some metal complexes can act as artificial proteases. In particular, the Lewis acid scandium(III) triflate has been shown to catalyze the cleavage of peptide bonds to serine and threonine residues. Therefore, we investigated if this compound can also be used for the cleavage of proteins. For this purpose, several single proteins, the 20S immune-proteasome (17 proteins), and the Universal Proteomics Standard UPS1 (48 proteins) were analyzed by MALDI–MS and/or LC–MS. A high cleavage specificity N-terminal to serine and threonine residues was observed, but also additional peptides with deviating cleavage specificity were found. Scandium(III) triflate can be a useful tool in protein analysis as no other reagent has been reported yet which showed cleavage specificity within proteins to serines and threonines.

• Bollineni, Ravi Chand; Koehler, Christian; Gislefoss, Randi Elin; Anonsen, Jan Haug & Thiede, Bernd (2018). Large-scale intact glycopeptide identification by Mascot database search. Scientific Reports. 8. doi: 10.1038/s41598-018-20331-2. Fulltekst i vitenarkiv Vis sammendrag

  Workflows capable of determining glycopeptides in large-scale are missing in the field of glycoproteomics. We present an approach for automated annotation of intact glycopeptide mass spectra. The steps in adopting the Mascot search engine for intact glycopeptide analysis included: (i) assigning one letter codes for monosaccharides, (ii) linearizing glycan sequences and (iii) preparing custom glycoprotein databases. Automated annotation of both N- and O-linked glycopeptides was proven using standard glycoproteins. In a large-scale study, a total of 257 glycoproteins containing 970 unique glycosylation sites and 3447 non-redundant N-linked glycopeptide variants were identified in 24 serum samples. Thus, a single tool was developed that collectively allows the (i) elucidation of N- and O-linked glycopeptide spectra, (ii) matching glycopeptides to known protein sequences, and (iii) high-throughput, batch-wise analysis of large-scale glycoproteomics data sets.

• Buehler, Ulrike; Schulenburg, Katharina; Yurugi, Hajime; Solman, Maja; Abankwa, Daniel & Ulges, Alexander [Vis alle 11 forfattere av denne artikkelen] (2018). Targeting prohibitins at the cell surface prevents Th17-mediated autoimmunity. EMBO Journal. ISSN 0261-4189. 37:e99429(16), s. 1–14. doi: 10.15252/embj.201899429. Fulltekst i vitenarkiv
• Skottvoll, Frøydis Sved; Berg, Henriette Engen; Bjørseth, Kamilla; Lund, Kaja; Roos, Norbert & Bekhradnia, Sara [Vis alle 13 forfattere av denne artikkelen] (2018). Ultracentrifugation versus kit exosome isolation: nanoLC-MS and other tools reveal similar performance biomarkers, but also contaminations. Future science OA. 5(1). doi: 10.4155/fsoa-2018-0088. Fulltekst i vitenarkiv Vis sammendrag

  Aim: For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared. Materials & methods: ‘Case study’ exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed. Results: Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLC–MS. Western blotting and nanoLC–MS also revealed negative exosome markers regarding both isolation approaches. Conclusion: The two isolation methods had an overall similar performance, but we hesitate to use the term ‘exosome isolation’ as impurities may be present with both isolation methods. NanoLC–MS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures.

• Tran, Trung; Bollineni, Ravi Chand; Koehler, Christian & Thiede, Bernd (2018). Absolute two-point quantification of proteins using dimethylated proteotypic peptides. The Analyst. ISSN 0003-2654. 143(18), s. 4359–4365. doi: 10.1039/c8an01081a. Fulltekst i vitenarkiv
• Aqrawi, Lara Adnan; Chen, Xiangjun; Jensen, Janicke Liaaen; Morthen, Mathias Kaurstad; Thiede, Bernd & Utheim, Øygunn Aass [Vis alle 10 forfattere av denne artikkelen] (2018). Severity of clinical dry eye manifestations influences protein expression in tear fluid of patients with primary Sjögren's syndrome. PLOS ONE. 13:e0205762(10), s. 1–14. doi: 10.1371/journal.pone.0205762. Fulltekst i vitenarkiv Vis sammendrag

  Ocular dryness is a characteristic feature of primary Sjögren’s syndrome (pSS). This may result in dry eye disease (DED), leading to damage of the ocular surface. Additional, non-invasive diagnostic techniques are needed when evaluating pSS patients. Hence, screening for disease-specific biomarkers in biological fluid could be promising. We have previously examined the proteome of tear fluid from pSS patients through Liquid chromatography-mass spectrometry (LC-MS), and conducted a thorough ocular evaluation of patients with pSS. In this study we further explored the association between dry eye manifestations and protein expression in tear fluid of pSS patients. Medical history of 27 patients and 32 healthy controls was gathered. Subjective complaints were registered through questionnaires. Objective findings including tear osmolarity, tear film break up time (TFBUT), Schirmer’s test, and ocular and corneal surface staining were also recorded. LC-MS was conducted formerly on tear fluid from all subjects in order to generate proteomic biomarker profiles. Scaffold was employed to analyse the LC-MS data for quantitative differences between patient and control groups, and the mean spectral counts were calculated for the five most upregulated proteins in relation to DED manifestations. Dysregulated cellular processes were identified in pSS patients using FunRichv3 enrichment analysis. The five most upregulated proteins previously identified in pSS patients were DNA (apurinic or apyrimidinic site) lyase (APEX1), thioredoxin-dependent peroxidase reductase (PRDX3), copine (CPNE1), aconitate hydratase (ACO2), and LIM domain only protein 7 (LMO7), in descending order. A significant increase in mean spectral counts for these proteins were observed in pSS patients with pathological DED manifestations compared to healthy controls (p<0.0001). Consequently, dysregulated cellular pathways involving innate and adaptive immunity were also detected. In conclusion, our observations suggest a relationship between presence of dry eye signs and upregulated proteins in tear fluid from patients with pSS. Further studies are needed in order to replicate the concepts explored and analyses performed in a greater cohort of pSS patients, where sensitivity and specificity of the methods conducted can also be verified further.

• Dugarte, Maria Eugenia Chollet; Andersen, Elisabeth; Skarpen, Ellen; Myklebust, Christiane Filion; Koehler, Christian & Morth, Jens Preben [Vis alle 12 forfattere av denne artikkelen] (2018). Factor VII deficiency: Unveiling the cellular and molecular mechanisms underlying Three model alterations of the enzyme catalytic domain. Biochimica et Biophysica Acta - Molecular Basis of Disease. ISSN 0925-4439. 1864(3), s. 660–667. doi: 10.1016/j.bbadis.2017.12.016. Fulltekst i vitenarkiv
• Pasovic, Lara; Utheim, Tor Paaske; Reppe, Sjur; Khan, Ayyad Ahmad Zartasht; Jackson, Catherine & Thiede, Bernd [Vis alle 9 forfattere av denne artikkelen] (2018). Improvement of storage medium for cultured human retinal pigment epithelial cells using factorial design. Scientific Reports. 8(1). doi: 10.1038/s41598-018-24121-8. Fulltekst i vitenarkiv Vis sammendrag

  Storage of human retinal pigment epithelium (hRPE) can contribute to the advancement of cell-based RPE replacement therapies. The present study aimed to improve the quality of stored hRPE cultures by identifying storage medium additives that, alone or in combination, contribute to enhancing cell viability while preserving morphology and phenotype. hRPE cells were cultured in the presence of the silk protein sericin until pigmentation. Cells were then stored for 10 days in storage medium plus sericin and either one of 46 diferent additives. Individual efects of each additive on cell viability were assessed using epifuorescence microscopy. Factorial design identifed promising additive combinations by extrapolating their individual efects. Supplementing the storage medium with sericin combined with adenosine, L-ascorbic acid and allopurinol resulted in the highest cell viability (98.6 ± 0.5%) after storage for three days, as measured by epifuorescence microscopy. Flow cytometry validated the fndings. Proteomics identifed 61 upregulated and 65 downregulated proteins in this storage group compared to the unstored control. Transmission electron microscopy demonstrated the presence of melanosomes after storage in the optimized medium. We conclude that the combination of adenosine, L-ascorbic acid, allopurinol and sericin in minimal essential medium preserves RPE pigmentation while maintaining cell viability during storage.

• Dukic, Aleksandra; Gerbaud, Pascal; Guibourdenche, Jean; Thiede, Bernd; Tasken, Kjetil & Pidoux, Guillaume (2018). Ezrin-anchored PKA phosphorylates serine 369 and 373 on connexin 43 to enhance gap junction assembly, communication, and cell fusion. Biochemical Journal. ISSN 0264-6021. 475(2), s. 455–476. doi: 10.1042/BCJ20170529. Fulltekst i vitenarkiv Vis sammendrag

  A limited number of human cells can fuse to form multinucleated syncytia. In the differentiation of human placenta, mononuclear cytotrophoblasts fuse to form an endocrinologically active, non-proliferative, multinucleated syncytium. This syncytium covers the placenta and manages the exchange of nutrients and gases between maternal and fetal circulation. We recently reported protein kinase A (PKA) to be part of a macromolecular signaling complex with ezrin and gap junction protein connexin 43 (Cx43) that provides cAMP-mediated control of gap junction communication. Here, we examined the associated phosphorylation events. Inhibition of PKA activity resulted in decreased Cx43 phosphorylation, which was associated with reduced trophoblast fusion and differentiation. In vitro studies using peptide arrays, together with mass spectrometry, pointed to serine 369 and 373 of Cx43 as the major PKA phosphorylation sites that increases gap junction assembly at the plasmalemma. A combination of knockdown and reconstitution experiments and gap-fluorescence loss in photobleaching assays with mutant Cx43 containing single or double phosphoserine-mimicking amino acid substitutions in putative PKA phosphorylation sites demonstrated that phosphorylation of S369 and S373 mediated gap junction communication, trophoblast differentiation, and cell fusion.

• Haatveit, Hanne Merethe; Wessel, Øystein; Markussen, Turhan; Lund, Morten; Thiede, Bernd & Nyman, Ingvild Berg [Vis alle 9 forfattere av denne artikkelen] (2017). Viral protein kinetics of piscine orthoreovirus infection in atlantic salmon blood cells. Viruses. 9:49(3), s. 1–18. doi: 10.3390/v9030049. Fulltekst i vitenarkiv Vis sammendrag

  Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1–2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection.

• Tran, Trung; Bollineni, Ravi Chand; Strozynski, Margarita Anna; Koehler, Christian & Thiede, Bernd (2017). Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches. Journal of Proteome Research. ISSN 1535-3893. 16(7), s. 2571–2578. doi: 10.1021/acs.jproteome.7b00126. Fulltekst i vitenarkiv
• Tran, Trung; Strozynski, Margarita Anna & Thiede, Bernd (2017). Quantitative phosphoproteome analysis of cisplatin-induced apoptosis in Jurkat T cells. Proteomics. ISSN 1615-9853. 17(11). doi: 10.1002/pmic.201600470. Fulltekst i vitenarkiv
• Grundt, Kirsten; Thiede, Bernd & Østvold, Anne Carine (2017). Identification of kinases phosphorylating 13 sites in the nuclear, DNA-binding protein NUCKS. Biochimica et Biophysica Acta - Proteins and Proteomics. ISSN 1570-9639. 1865(3), s. 359–369. doi: 10.1016/j.bbapap.2016.12.009. Fulltekst i vitenarkiv
• Barfeld, Stefan Jörg; Urbanucci, Alfonso; Itkonen, Harri; Fazli, Ladan; Hicks, Jessica L & Thiede, Bernd [Vis alle 10 forfattere av denne artikkelen] (2017). c-Myc antagonises the transcriptional activity of the androgen receptor in prostate cancer affecting key gene networks. EBioMedicine. 18, s. 83–93. doi: 10.1016/j.ebiom.2017.04.006. Fulltekst i vitenarkiv Vis sammendrag

  Prostate cancer (PCa) is the most common non-cutaneous cancer in men. The androgen receptor (AR), a ligand-activated transcription factor, constitutes the main drug target for advanced cases of the disease. However, a variety of other transcription factors and signaling networks have been shown to be altered in patients and to influence AR activity. Amongst these, the oncogenic transcription factor c-Myc has been studied extensively in multiple malignancies and elevated protein levels of c-Myc are commonly observed in PCa. Its impact on AR activity, however, remains elusive. In this study, we assessed the impact of c-Myc overexpression on AR activity and transcriptional output in a PCa cell line model and validated the antagonistic effect of c-MYC on AR-targets in patient samples. We found that c-Myc overexpression partially reprogrammed AR chromatin occupancy and was associated with altered histone marks distribution, most notably H3K4me1 and H3K27me3. We found c-Myc and the AR co-occupy a substantial number of binding sites and these exhibited enhancer-like characteristics. Interestingly, c-Myc overexpression antagonised clinically relevant AR target genes. Therefore, as an example, we validated the antagonistic relationship between c-Myc and two AR target genes, KLK3 (alias PSA, prostate specific antigen), and Glycine N-Methyltransferase (GNMT), in patient samples. Our findings provide unbiased evidence that MYC overexpression deregulates the AR transcriptional program, which is thought to be a driving force in PCa.

• Stakkestad, Øystein; Lyngstadaas, Ståle Petter; Thiede, Bernd; Vondrasesek, Jiri; Skålhegg, Bjørn Steen & Reseland, Janne Elin (2017). Phosphorylation Modulates Ameloblastin Self-assembly and Ca2+ Binding. Frontiers in Physiology. 8. doi: 10.3389/fphys.2017.00531. Fulltekst i vitenarkiv Vis sammendrag

  Ameloblastin (AMBN), an important component of the self-assembled enamel extra cellular matrix, contains several in silico predicted phosphorylation sites. However, to what extent these sites actually are phosphorylated and the possible effects of such post-translational modifications are still largely unknown. Here we report on in vitro experiments aimed at investigating what sites in AMBN are phosphorylated by casein kinase 2 (CK2) and protein kinase A (PKA) and the impact such phosphorylation has on self-assembly and calcium binding. All predicted sites in AMBN can be phosphorylated by CK2 and/or PKA. The experiments show that phosphorylation, especially in the exon 5 derived part of the molecule, is inversely correlated with AMBN self-assembly. These results support earlier findings suggesting that AMBN self-assembly is mostly dependent on the exon 5 encoded region of the AMBN gene. Phosphorylation was significantly more efficient when the AMBN molecules were in solution and not present as supramolecular assemblies, suggesting that post-translational modification of AMBN must take place before the enamel matrix molecules self-assemble inside the ameloblast cell. Moreover, phosphorylation of exon 5, and the consequent reduction in self-assembly, seem to reduce the calcium binding capacity of AMBN suggesting that post-translational modification of AMBN also can be involved in control of free Ca2+ during enamel extra cellular matrix biomineralization. Finally, it is speculated that phosphorylation can provide a functional crossroad for AMBN either to be phosphorylated and act as monomeric signal molecule during early odontogenesis and bone formation, or escape phosphorylation to be subsequently secreted as supramolecular assemblies that partake in enamel matrix structure and mineralization.

• Koehler, Christian; Bollineni, Ravi Chand & Thiede, Bernd (2017). Application of the half decimal place rule to increase the peptide identification rate. Rapid Communications in Mass Spectrometry. ISSN 0951-4198. 31(2), s. 227–233. doi: 10.1002/rcm.7780. Fulltekst i vitenarkiv Vis sammendrag

  Rationale: Many MS2 spectra in bottom-up proteomics experiments remain unassigned. To improve proteome coverage, we applied the half decimal place rule (HDPR) to remove non-peptidic molecules. The HDPR considers the ratio of the digits after the decimal point to the full molecular mass and results in a relatively small permitted mass window for most peptides. Methods: First, the HDPR mass filter was calculated for the human and other proteomes. Subsequently, the HDPR was applied to three technical replicates of an in-solution tryptic digest of HeLa cells which were analysed by liquid chromatography/mass spectrometry (LC/MS) using a quadrupole-orbitrap mass spectrometer (Q Exactive). In addition, the same sample was analysed three times with a fixed exclusion list. The exclusion list was based on only choosing doubly charged ions for fragmentation. Results: The peptide spectrum match (PSM) rate increased by 2–4% applying HDPR filters from 0.1–0.25 Da and 75–150 ppm, respectively. Excluding all MS2 events by applying an HDPR filter of doubly charged ions, we were able to improve PSMs by 0.9% and the PSM rate by 2.5%. Conclusions: An algorithm to filter precursors based on the HDPR was established to improve the targeting of the acquisition of MS2 spectra in data-dependent acquisition (DDA) experiments. According to our data, a total gain of PSMs of 1–5% might be achievable if the HPDR filter would already be applied during MS data acquisition. Copyright © 2016 John Wiley & Sons, Ltd. The final version of this research has been published in Rapid Communications in Mass Spectrometry. © 2016 Wiley

• Soop, Graciela Lopez; Rønningen, Torunn; Rogala, Agnieszka; Richartz, Nina; Blomhoff, Heidi Kiil & Thiede, Bernd [Vis alle 8 forfattere av denne artikkelen] (2017). AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint. Cell Cycle. ISSN 1538-4101. 16(10), s. 947–956. doi: 10.1080/15384101.2017.1310350. Fulltekst i vitenarkiv Vis sammendrag

  Faithful chromosome segregation during mitosis relies on a proofreading mechanism that monitors proper kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) is based on the concerted action of numerous components that maintain a repressive signal inhibiting transition into anaphase until all chromosomes are attached. Here we show that A-Kinase Anchoring Protein 95 (AKAP95) is necessary for proper SAC function. AKAP95-depleted HeLa cells show micronuclei formed from lagging chromosomes at mitosis. Using a BioID proximity-based proteomic screen, we identify the nuclear pore complex protein TPR as a novel AKAP95 binding partner. We show interaction between AKAP95 and TPR in mitosis, and an AKAP95-dependent enrichment of TPR in the spindle microtubule area in metaphase, then later in the spindle midzone area. AKAP95-depleted cells display faster prometaphase to anaphase transition, escape from nocodazole-induced mitotic arrest and show a partial delocalization from kinetochores of the SAC component MAD1. Our results demonstrate an involvement of AKAP95 in proper SAC function likely through its interaction with TPR.

• Aqrawi, Lara Adnan; Galtung, Hilde; Vestad, Beate; Øvstebø, Reidun; Thiede, Bernd & Rusthen, Shermin [Vis alle 13 forfattere av denne artikkelen] (2017). Identification of potential saliva and tear biomarkers in primary Sjögren’s syndrome, utilising the extraction of extracellular vesicles and proteomics analysis. Arthritis Research & Therapy. ISSN 1478-6354. 19(1). doi: 10.1186/s13075-017-1228-x. Fulltekst i vitenarkiv Vis sammendrag

  Background There is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sjögren’s syndrome (pSS) patients. Incorporation of additional diagnostics involving screening for disease-specific biomarkers in biological fluid is a promising concept that requires further investigation. In the current study we aimed to explore novel disease biomarkers in saliva and tears from pSS patients. Methods Liquid chromatography-mass spectrometry (LC-MS) was performed on stimulated whole saliva and tears from 27 pSS patients and 32 healthy controls, and salivary and tear proteomic biomarker profiles were generated. LC-MS was also combined with size exclusion chromatography to isolate extracellular vesicles (EVs) from both fluids. Nanoparticle tracking analysis was conducted on joint fractions from the saliva and tears to determine size distribution and concentration of EVs. Further EV characterisation was performed by immunoaffinity capture of CD9-positive EVs using magnetic beads, detected by flow cytometry. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold, and the proteins were further analysed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), for gene ontology overrepresentation, and the Search Tool for the Retrieval of Interacting Genes/Proteins for protein-protein interaction network analysis. Results Upregulation of proteins involved in innate immunity (LCN2), cell signalling (CALM) and wound repair (GRN and CALML5) were detected in saliva in pSS. Saliva EVs also displayed biomarkers critical for activation of the innate immune system (SIRPA and LSP1) and adipocyte differentiation (APMAP). Tear analysis indicated overexpression of proteins involved in TNF-α signalling (CPNE1) and B cell survival (PRDX3). Moreover, neutrophil gelatinase-associated lipocalin was upregulated in saliva and tears in pSS. Consistently, DAVID analysis demonstrated pathways of the adaptive immune response in saliva, of cellular component assembly for saliva EVs, and of metabolism and protein folding in tears in pSS patients. Conclusions LC-MS of saliva and tears from pSS patients, solely and in combination with size-exclusion chromatography allowed screening for possible novel biomarkers encompassing both salivary and lacrimal disease target organs. This approach could provide additional diagnostic accuracy in pSS, and could possibly also be applied for staging and monitoring the disease.

• Imre, Gergely; Berthelet, Jean; Heering, Jan; Kehrloesser, Sebastian; Melzer, Inga Maria & Lee, Byung Il [Vis alle 9 forfattere av denne artikkelen] (2017). Apoptosis inhibitor 5 is an endogenous inhibitor of caspase-2. EMBO Reports. ISSN 1469-221X. 18(5), s. 733–744. doi: 10.15252/embr.201643744. Fulltekst i vitenarkiv Vis sammendrag

  Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase‐2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase‐2 also regulates autophagy, genomic stability and ageing. Caspase‐2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase‐2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase‐2 and impedes dimerization and activation of caspase‐2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase‐2. Depletion of endogenous API5 leads to an increase in caspase‐2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase‐2‐dependent apoptotic cell death. These results establish API5/AAC‐11 as a direct inhibitor of caspase‐2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.

• Fiorito, Elisa; Sharma, Yogita; Gilfillan, Siv; Wang, Shixiong; Singh, Sachin Kumar & Somisetty, Venkata Satheesh [Vis alle 11 forfattere av denne artikkelen] (2016). CTCF modulates Estrogen Receptor function through specific chromatin and nuclear matrix interactions. Nucleic Acids Research (NAR). ISSN 0305-1048. 44, s. 10588–10602. doi: 10.1093/nar/gkw785. Fulltekst i vitenarkiv
• Gerner, Lisa; Munack, Steffi; Temmerman, Koen; Lawrence-Dörner, Ann-Marie; Besir, Huseyin & Wilmanns, Matthias [Vis alle 10 forfattere av denne artikkelen] (2016). Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli. Data in Brief. 8, s. 733–740. doi: 10.1016/j.dib.2016.06.033. Fulltekst i vitenarkiv
• Olsen, Christel Moræus; Markussen, Turhan; Thiede, Bernd & Rimstad, Espen (2016). Infectious Salmon Anaemia Virus (ISAV) RNA Binding Protein Encoded by Segment 8 ORF2 and Its Interaction with ISAV and Intracellular Proteins. Viruses. 8(2). doi: 10.3390/v8020052. Fulltekst i vitenarkiv Vis sammendrag

  Infectious salmon anaemia virus (ISAV) is an orthomyxovirus infecting salmonid fish. The virus is adapted to low temperature and has a replication optimum between 10–15 °C. In this study the subcellular localization and protein interactions for the protein encoded by the largest open reading frame of gene segment 8 (s8ORF2) were investigated. In ISAV infected cells the s8ORF2 protein was found mainly in the cytosol but a minor fraction of cells expressed the protein in the nucleus as well. Green fluorescent protein-tagged s8ORF2 did not leak out of the cell when the plasma membrane was permeabilized, suggesting interactions with intracellular structural components. The s8ORF2 protein exists both as monomer and homodimer, and co-immunoprecipitation experiments strongly suggests it binds to the ISAV fusion-, nucleo- and matrix proteins. Two versions of s8ORF2 were detected with apparent molecular weights of 24–26 and 35 kDa in lysates of infected cells. The 35 kDa type is an early viral protein while the smaller version appears during the later phases of infection. The 24–26 kDa type was also the predominant form in viral particles. The s8ORF2 protein has previously been shown to bind RNA and interfere with interferon induction and signaling. Here we found that a fraction of the s8ORF2 protein pool in infected cells is likely to be conjugated to the interferon stimulated gene 15 (ISG15) and ubiquitin. Furthermore, several endogenous proteins pulled down by the s8ORF2 protein were identified by liquid chromatography mass spectrometry (LC-MS).

• Gerner, Lisa; Munack, Steffi; Temmerman, Koen; Lawrence-Dörner, Ann-Marie; Besir, Huseyin & Wilmanns, Matthias [Vis alle 10 forfattere av denne artikkelen] (2016). Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2. Biochemical and Biophysical Research Communications - BBRC. ISSN 0006-291X. 476(2), s. 102–107. doi: 10.1016/j.bbrc.2016.05.045. Fulltekst i vitenarkiv
• Arntzen, Magnus Øverlie; Boddie, Paul; Frick, Rahel; Koehler, Christian & Thiede, Bernd (2015). Consolidation of proteomics data in the Cancer proteomics database. Proteomics. ISSN 1615-9853. 15(22), s. 3765–3771. doi: 10.1002/pmic.201500144. Fulltekst i vitenarkiv
• Serguienko, Anastassia; Grad, Iwona; Wennerstrøm, Anna; Meza, Leonardo Zepeda; Thiede, Bernd & Stratford, Eva Wessel [Vis alle 8 forfattere av denne artikkelen] (2015). Metabolic reprogramming of metastatic breast cancer and melanoma by Let-7a microRNA. OncoTarget. 6(4), s. 2451–2465. doi: 10.18632/oncotarget.3235. Fulltekst i vitenarkiv
• Villa, Oscar; Brookes, Steven J; Thiede, Bernd; Heijl, Lars; Lyngstadaas, Ståle Petter & Reseland, Janne Elin (2015). Subfractions of enamel matrix derivative differentially influence cytokine secretion from human oral fibroblasts. Journal of Tissue Engineering. 6, s. 1–11. doi: 10.1177/2041731415575857. Fulltekst i vitenarkiv Vis sammendrag

  Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase of the treatment of periodontal defects. Our main goal was to analyze the bioactivity of different molecular weight fractions of enamel matrix derivative. Enamel matrix derivative, a complex mixture of proteins, was separated into 13 fractions using size-exclusion chromatography and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–electrospray ionization–tandem mass spectrometry. Human periodontal ligament fibroblasts were treated with either enamel matrix derivative or the different fractions. Proliferation and cytokine secretion to the cell culture medium were measured and compared to untreated cells. The liquid chromatography–electrospray ionization–tandem mass spectrometry analyses revealed that the most abundant peptides were amelogenin and leucine-rich amelogenin peptide related. The fractions containing proteins above 20 kDa induced an increase in vascular endothelial growth factor and interleukin-6 secretion, whereas lower molecular weight fractions enhanced proliferation and secretion of interleukin-8 and monocyte chemoattractant protein-1 and reduced interleukin-4 release. The various molecular components in the enamel matrix derivative formulation might contribute to reported effects on tissue regeneration through their influence on vascularization, the immune response, and chemotaxis.

• Bousquet, Paula; Sandvik, Joe Alexander; Arntzen, Magnus Øverlie; Edin, Nina Frederike J; Christoffersen, Stine & Krengel, Ute [Vis alle 8 forfattere av denne artikkelen] (2015). Hypoxia Strongly Affects Mitochondrial Ribosomal Proteins and Translocases, as Shown by Quantitative Proteomics of HeLa Cells. International Journal of Proteomics. ISSN 2090-2174. doi: 10.1155/2015/678527. Fulltekst i vitenarkiv
• Chymkowitch, Pierre; P, Aurélie Nguéa; Aanes, Håvard; Koehler, Christian; Thiede, Bernd & Lorenz, Susanne [Vis alle 9 forfattere av denne artikkelen] (2015). Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes. Genome Research. ISSN 1088-9051. 25(6), s. 897–906. doi: 10.1101/gr.185793.114. Fulltekst i vitenarkiv
• Albrecht, Elke; norheim, frode; Thiede, Bernd; Lee, Sindre; Holen, Torgeir & Ohashi,, Tomoo [Vis alle 12 forfattere av denne artikkelen] (2015). Irisin – a myth rather than an exercise-inducible myokine. Scientific Reports. doi: 10.1038/srep08889. Fulltekst i vitenarkiv
• Bollineni, Ravi Chand; Guldvik, Ingrid Jenny; Grönberg, Henrik; Wiklund, Fredrik; Mills, Ian Geoffrey & Thiede, Bernd (2015). A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate. The Analyst. ISSN 0003-2654. 140(24), s. 8109–8117. doi: 10.1039/c5an01560j. Fulltekst i vitenarkiv
• Øverbye, Anders; Skotland, Tore; Koehler, Christian; Thiede, Bernd; Seierstad, Therese & Berge, Viktor [Vis alle 8 forfattere av denne artikkelen] (2015). Identification of prostate cancer biomarkers in urinary exosomes. OncoTarget. 6(30), s. 30357–30376. doi: 10.18632/oncotarget.4851. Fulltekst i vitenarkiv
• Albrecht, Elke; Norheim, Frode; Thiede, Bernd; Holen, Torgeir; Ohashi, Tomoo & Schering, Lisa [Vis alle 12 forfattere av denne artikkelen] (2015). Irisin - A myth rather than an exercise-inducible myokine. Scientific Reports. 5. doi: 10.1038/srep08889. Fulltekst i vitenarkiv

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• Dugarte, Maria Eugenia Chollet; Harrison, Sean; Baumgarten, Saphira Felicitas; Morena-Barrio, Maria Eugenia de la; Bravo-Pérez, Carlos & Mowinckel, Marie-Christine [Vis alle 12 forfattere av denne artikkelen] (2022). Human iPSC-derived hepatic organoids – a model for blood coagulation research and therapy. Human Gene Therapy. ISSN 1043-0342. doi: 10.1089/hum.2022.29225.abstracts. Fulltekst i vitenarkiv
• Hanson, Ingunn; Pitman, Kathinka Elinor; Thiede, Bernd; Cai, Yiqing & Edin, Nina Frederike Jeppesen (2021). Proteomic Analysis of Low Dose-Rate Primed Cells and Extracellular Exosomes. Fulltekst i vitenarkiv
• Bakketeig, Eris Aas; Olsen, Christine; Berg, Henriette Engen; Nyman, Tuula Anneli; Thiede, Bernd & Lundanes, Elsa [Vis alle 8 forfattere av denne artikkelen] (2021). Optimization of concatenation of fractions in comprehensive two dimensional liquid chromatography bottom-up proteomics. Fulltekst i vitenarkiv Vis sammendrag

  Two dimensional liquid chromatography (2D-LC) has long been a commonly used technique in bottom-up proteomics, and is as relevant today as ever [1, 2]. To maximize the number of protein identifications, analytical chemists have strived to obtain 2D-LC systems with a high degree of orthogonality, which is needed to reach the required high peak capacities. A system utilizing reversed-phase chromatography (RP) in both column dimensions with a large difference in pH has proven to yield a higher peak capacity for peptides, even with a lower degree of orthogonality compared to other combinations [3]. The present study is based on Reubsaet, et al. [4]. The study is a comprehensive offline RPRP 2D-LC system with high pH (>9) in the first dimension and low pH (3) in the second dimension. The method’s purpose is to maximise protein identifications from one trypsinated protein sample. To achieve maximum identifications, Reubsaet incorporated the concept of concatenation of fractions. In practice, concatenation is applied through collecting the column output from the first dimension in eight numbered vials, where the 1st, 9th, 17th, … fractions are collected in the first vial, the 2nd, 10th, 18th, … are collected in the second, etc. [4]. In the present study, the fractionation scheme is examined by changing the total number of vials in which samples are concatenated, the number of fractions in each vial, and the collection time for each fraction. As the method demands high precision and is very timeconsuming, automatization is practically essential. Automatization is achieved with the spider fractionation scheme, proposed and shown by Kulak, et al.[5]. Here, a 10-port injector connects the first dimension column to each sample vial, and switches between them automatically, reducing the strain on the operator drastically, while removing human errors. The optimization of the concatenation will lead to higher numbers of identified proteins in proteomics.

• Dugarte, Maria Eugenia Chollet; andersen, elisabeth; Morena-Barrio, Maria Eugenia de la; Bravo-Perez, Carlos; Mowinckel, Marie-Christine & Myklebust, Christiane Fillion [Vis alle 12 forfattere av denne artikkelen] (2020). Characterizing Coagulation FVII from iPSC-Hepatocytes-like Cells: Setting the Basis for Cell Therapy Development. Blood. ISSN 0006-4971. 136. doi: 10.1182/blood-2020-139211. Fulltekst i vitenarkiv
• Hanson, Ingunn; Edin, Nina Frederike J; Thiede, Bernd & Altanerova, Ursula (2020). Communication of radioprotective efects by TGF-β3. Fulltekst i vitenarkiv
• Berg, Henriette Engen; Halldórsson, Skarphéðinn; Thiede, Bernd; Vik-Mo, Einar O.; Lundanes, Elsa & Wilson, Steven Ray Haakon (2020). Micro pillar array columns for comprehensive proteomics. Fulltekst i vitenarkiv Vis sammendrag

  Pillar array nano liquid chromatography columns can potentially replace packed nano liquid chromatography (nLC) columns for analyses of complex samples due to high chromatographic performance and low backpressure. In our study, pillar array columns are used for the detection of potential biomarkers in Glioblastoma Multiforme (GBM), which is the most aggressive form of brain cancer in adults. Pillar array columns consist of several micropillars etched into silicon wafers using lithographic micromachining. The interest of these columns in nLC format has increased as an option to packed nLC columns as the chromatographic efficiency could outperform nLC columns packed with sub-2 µm particles while maintaining the sensitivity [1]. In contrast to packed columns, where eddy dispersion is the main source of band broadening due to irregular flow paths between the particles, the perfectly ordered pillars in pillar array columns eliminate the eddy dispersion to a minimum. The low backpressure of pillar array columns compared to that of packed columns also permits the use of exceptionally long columns, increasing the chromatographic performance further. Using pillar array columns, plate numbers over 1 million and reduced plate heights (h) even <1 µm have been achieved [2-4]. High chromatographic performance is especially important in comprehensive proteomics. Pre-fractionation (using e.g. gel electrophoresis) is often added in proteomic protocols to increase the throughput [5]. However, performing pre-fractionation can be time-consuming in addition to the highly increased instrument time required. Recently, a micro-Chip pillar array nLC column (µPACTM) compatible with standard nLC instrumentation has been commercialized (Figure 1) [6]. Our question was; could the use of µPAC nLC columns reduce the need for pre-fractionation in comprehensive proteomics of GBM biopsies?

• Hanson, Ingunn; Altanerova, Ursula; Thiede, Bernd & Edin, Nina Frederike J (2020). Radioprotector TGF-β3 is secreted in exosomes and activated by LDR priming. Fulltekst i vitenarkiv Vis sammendrag

  The biological effects of ionizing radiation in the low-dose region is important for diagnostic imaging, healthy tissue in radiation therapy and environmental radiation. For a majority of tested cell lines, the low dose hyper-radiosensitive (HRS) response has been shown to be default. We have previously shown that cells primed with a low dose-rate (⁓0.3 Gy/h) ionizing radiation permanently loses the HRS response. We have also shown that the transforming growth factor (TGF)-β3 is responsible for this effect. Here, we investigated the secretion mechanism of TGF-β3 from LDR primed cells, and theorize regarding the way it is activated. Exosomes were isolated from LDR primed cells, and were shown to remove HRS in reporter cells when co-cultured with these. Adding a TGF-β3 inhibitor to the medium restored the HRS response in reporter cells, confirming that TGF-β3 was the factor in the exosomes responsible for removing HRS. Mass spectrometry was performed on the content of exosomes from primed and control cells. Proteomic analysis confirmed the presence of exosome-specific proteins in exosomes from primed and control groups. TGF-β3 along with latency-associated protein (LAP), the protein that TGF-β3 is bound to in its inactive form, was present in similar amounts in both groups, indicating that TGF-β3 was secreted in its inactive form, regardless of LDR priming. Three proteins were significantly upregulated in the primed exosomes. These proteins will be investigated further in future experiments. We conclude that TGF-β3 is secreted in its inactive form in exosomes from LDR primed and unprimed cells, and that the priming is responsible for its activation. We will further investigate three significantly upregulated proteins with regards to their role in the activation of TGF-β3.

• Bollineni, Ravi Chand; Koehler, Christian; Gislefoss, Randi Elin; Anonsen, Jan Haug & Thiede, Bernd (2018). Author Correction: Large-scale intact glycopeptide identification by Mascot database search. Scientific Reports. 8. doi: 10.1038/s41598-018-28147-w. Fulltekst i vitenarkiv
• Eriksen, Gunnar Sundstøl; Thiede, Bernd & Solhaug, Anita (2018). Effects of an emerging Fusarium mycotoxin on hepatocytes. Fulltekst i vitenarkiv
• Khan, Ayyad Ahmad Zartasht; Jackson, Catherine; Reppe, Sjur; Utheim, Tor Paaske; Olstad, Ole Kristoffer & Thiede, Bernd [Vis alle 7 forfattere av denne artikkelen] (2018). Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelium is Associated with Elevated Levels of Hydrogen Peroxide. Fulltekst i vitenarkiv
• Berg, Henriette Engen; Skottvoll, Frøydis Sved; Bjørseth, Kamilla; Sæterdal, Kristina Erikstad; Brandtzaeg, Ole Kristian & Vehus, Tore [Vis alle 11 forfattere av denne artikkelen] (2018). Development of nanoLC column technology for proteomic studies. Fulltekst i vitenarkiv Vis sammendrag

  Background NanoLC has emerged as a major technique in proteomics due to improved sensitivity, which enables analysis of small sample volumes and low concentrations. In our group (Bioanalytical chemistry, BACH) we focus on the development of column technology, including porous layer open tubular (PLOT) columns and self-packing of columns for proteomic applications. Methods The PLOT columns were used for both on-line digestion (enzyme reactors) and separations (10 µm ID, PS-DVB with ODS). Enzyme reactors (20 µm ID and multi-PLOT columns (~8 µm ID)) were immobilized with trypsin and Lys-C for protein digestion. We also compared self-packed columns of 50 µm ID (2.6 µm C18 core-shell particles) to a commercial column (75 µm ID). The columns were used for detection of CYP27a1 in MDA-MB-231 (breast cancer) cells, and biomarker candidates in MDA-MB-231 and glioblastoma multiforme (brain cancer) exosomes. For exosome isolation (cell culture medium), ultracentrifugation (“golden standard”) was compared to a Total Exosome Isolation Reagent from Thermo Fisher regarding protein yield and purity. Results Our PLOT columns were successfully employed for proteins, metabolites and peptides with attogram detection limits and peak capacities around 250 [1]. The enzyme reactors provided sufficient digestion in ~5 minutes compared to overnight digestions (common in off-line protocols) [2]. Furthermore, a quantitative method of the neurotoxin ricin was developed using the multi-PLOT to reduce manual sample handling [3] . The self-packed columns obtained peak capacities comparable to the commercial nanoLC column for peptide separations [4]. The self-packed columns were used for the targeted detection of CYP27A1 (potential breast cancer biomarker) in MDA-MB-231 cells. In the recent exosome study, exosomes were present and biomarker candidates were identified. However, the characterization techniques are in our hands not satisfactory. Conclusions We have successfully developed sensitive nanoLC column formats for proteomic applications.

• Skottvoll, Frøydis Sved; Lund, Kaja; Røberg-Larsen, Hanne; Berg, Henriette Sjånes; Thiede, Bernd & Lundanes, Elsa [Vis alle 7 forfattere av denne artikkelen] (2017). Diagnostic potential of exosomes: The purification issue. Fulltekst i vitenarkiv
• López-Soop, Graciela; Rønningen, Torunn; Rogala, Agnieszka; Richartz, Nina; Blomhoff, Heidi Kiil & Thiede, Bernd [Vis alle 8 forfattere av denne artikkelen] (2017). AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint. Cell Cycle. ISSN 1538-4101. 16(10), s. 947–956. doi: 10.1080/15384101.2017.1310350. Fulltekst i vitenarkiv
• Jungblut, Peter R.; Thiede, Bernd & Schluter, H (2016). Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts. Journal of Proteomics. ISSN 1874-3919. 134, s. 1–4. doi: 10.1016/j.jprot.2016.01.012. Fulltekst i vitenarkiv
• Jensen, Janicke Liaaen; Utheim, Øygunn Aass; Chen, Xiangjun; Thiede, Bernd; Aqrawi, Lara Adnan & Rusthen, Shermin [Vis alle 9 forfattere av denne artikkelen] (2016). Searching for Disease Biomarkers for Primary Sjögren’s Syndrome Based on Oral and Ocular Patient Examinations. Fulltekst i vitenarkiv
• Soop, Graciela Lopez; Reiner, Andrew Henry; Thiede, Bernd; Collas, Philippe & Küntziger, Thomas Michel (2015). Regulation of Normal Mitotic Progression by A-kinase Anchoring Protein 95 (AKAP95). Fulltekst i vitenarkiv
• Berg, Henriette Engen; Wilson, Steven Ray Haakon; Lundanes, Elsa; Thiede, Bernd & Vik-Mo, Einar Osland (2022). Exploring miniaturized chromatography formats and sample preparation methods for shotgun proteomics in limited biological samples. Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Oslo. Fulltekst i vitenarkiv Vis sammendrag

  A significant challenge in medical diagnostics is the development of simple but efficient tools for the detection/quantification of several biomarkers simultaneously using non-invasive sampling techniques. In this regard, the analysis of proteins (proteomics) is essential for understanding cellular processes and biomarker discovery. However, proteins vary greatly in terms of concentration levels and chemical properties in biological materials. Further, low sample sizes of modern biological models (e.g., patient-derived cell cultures, exosomes, and organoids) remain a big analytical challenge. The present work has focused on the brain cancer glioblastoma, which is in great need of increased knowledge and non-invasive sampling techniques. In addition, human organoids, which could act as a future in vitro model for disease modeling and personalized medicine, have been investigated. We have used high-resolution mass spectrometry for protein identification, exploring a selection of miniaturized liquid chromatography formats (for separation) and sample preparation techniques. By implementing these techniques, we have been able to study exosomes, 2D/3D cell cultures, and organoids, identifying over 6300 proteins in a single run using less than 5 µg of protein. The work has provided important insight into the possibilities and challenges of several novel models. It represents a development toward deeper proteomic profiling focusing on maintaining a high protein yield and time efficiency.

• Bakketeig, Eris Aas; Wilson, Steven Ray Haakon; Lundanes, Elsa; Berg, Henriette Engen; Olsen, Christine & Reubsaet, Leon [Vis alle 8 forfattere av denne artikkelen] (2021). The setup and optimization of a complete global proteomics two-dimensional liquid chromatography system, using Spider Fractionation. Universitetet i Oslo. Fulltekst i vitenarkiv

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