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• 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. Full text in Research Archive Show summary

  (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. In Marcus, Katrin; Eisenacher, Martin & Sitek, Barbara (Ed.), Quantitative Methods in Proteomics. Humana Press. ISSN 9781071610237. p. 133–144. doi: 10.1007/978-1-0716-1024-4_10. Full text in Research Archive Show summary

  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.

• Pizzi, Eleonora; Halvorsen, Trine Grønhaug; Koehler, Christian & Reubsaet, Leon (2021). Next-Generation Dried Blood Spot Samplers for Protein Analysis: Describing Trypsin-Modified Smart Sampling Paper. Separations. 8(5). doi: 10.3390/separations8050066. Full text in Research Archive Show summary

  This paper describes smart sampling paper to be used for bottom-up protein analysis. Four different manners to immobilize trypsin on cellulose were evaluated. Untreated paper, potassium-periodate-functionalized paper (with and without post-immobilization reduction) and 2-hydroxyethyl methacrylate (HEMA)/2-vinyl-4,4-dimethylazlactone (VDM)-functionalized paper were all used to immobilize trypsin. For the evaluation, Coomassie Brilliant Blue staining of proteins on paper and the BAEE trypsin activity assay needed to be modified. These methods allowed, together with data from mass spectrometric analysis of cytochrome C digestions, us to acquire fundamental insight into protein binding, and trypsin action and activity on paper. All functionalized discs bind more protein than the untreated discs. Protein binding to functionalized discs is based on both adsorption and covalent binding. Trypsin immobilized on potassium-periodate-functionalized discs exhibits the highest trypsin activity when using cytochrome C as substrate. It is proven that it is trypsin attached to paper (and not desorbed trypsin) which is responsible for the enzyme activity. The use of discs on complex biological samples shows that all functionalized discs are able to digest diluted serum; for the best-performing disc, HEMA-VDM functionalized, up to 200 high-confidence proteins are qualified, showing its potential.

• 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, p. 61–66. doi: 10.1007/s00775-019-01733-7. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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.

• 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), p. 4359–4365. doi: 10.1039/c8an01081a. Full text in Research Archive
• Dugarte, Maria Eugenia Chollet; Andersen, Elisabeth; Skarpen, Ellen; Myklebust, Christiane Filion; Koehler, Christian & Morth, Jens Preben [Show all 12 contributors for this article] (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), p. 660–667. doi: 10.1016/j.bbadis.2017.12.016. Full text in Research Archive
• 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), p. 2571–2578. doi: 10.1021/acs.jproteome.7b00126. Full text in Research Archive
• 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), p. 227–233. doi: 10.1002/rcm.7780. Full text in Research Archive Show summary

  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

• 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), p. 3765–3771. doi: 10.1002/pmic.201500144. Full text in Research Archive
• Chymkowitch, Pierre; P, Aurélie Nguéa; Aanes, Håvard; Koehler, Christian; Thiede, Bernd & Lorenz, Susanne [Show all 9 contributors for this article] (2015). Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes. Genome Research. ISSN 1088-9051. 25(6), p. 897–906. doi: 10.1101/gr.185793.114. Full text in Research Archive
• Øverbye, Anders; Skotland, Tore; Koehler, Christian; Thiede, Bernd; Seierstad, Therese & Berge, Viktor [Show all 8 contributors for this article] (2015). Identification of prostate cancer biomarkers in urinary exosomes. OncoTarget. 6(30), p. 30357–30376. doi: 10.18632/oncotarget.4851. Full text in Research Archive
• Koehler, Christian; Arntzen, Magnus Øverlie & Thiede, Bernd (2015). The impact of carbon-13 and deuterium on relative quantification of proteins using stable isotope diethyl labeling. Rapid Communications in Mass Spectrometry. ISSN 0951-4198. 29(9), p. 830–836. doi: 10.1002/rcm.7170. Full text in Research Archive
• Koehler, Christian; Arntzen, Magnus; Souza, Gustavo Antonio De & Thiede, Bernd (2013). An Approach for Triplex-Isobaric Peptide Termini Labeling (Triplex-IPTL). Analytical Chemistry. ISSN 0003-2700. 85(4), p. 2478–2485. doi: 10.1021/ac3035508. Full text in Research Archive
• Størvold, Gro Leite; Landskron, Johannes; Strozynski, Margarita Anna; Arntzen, Magnus; Koehler, Christian & Kalland, Maria Elisabeth [Show all 8 contributors for this article] (2013). Quantitative profiling of tyrosine phosphorylation revealed changes in the activity of the T cell receptor signaling pathway upon cisplatin-induced apoptosis. Journal of Proteomics. ISSN 1874-3919. 91, p. 344–357. doi: 10.1016/j.jprot.2013.07.019. Full text in Research Archive
• Thiede, Bernd; Koehler, Christian; Strozynski, Margarita Anna; Treumann, Achim; Stein, Robert & Zimny-Arndt, Ursula [Show all 8 contributors for this article] (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 Chromatograpohy Coupled to an LTQ-OrbitrapMass Spectrometer. Molecular & Cellular Proteomics. ISSN 1535-9476. 12(2), p. 529–538. doi: 10.1074/mcp.M112.019372. Full text in Research Archive
• Koehler, Christian; Arntzen, Magnus; Treumann, Achim & Thiede, Bernd (2012). A Rapid Approach for Isobaric Peptide Termini Labeling. Methods in molecular biology. ISSN 1064-3745. 893, p. 129–141. doi: 10.1007/978-1-61779-885-6_10. Full text in Research Archive
• Koehler, Christian; Arntzen, Magnus; Treumann, Achim & Thiede, Bernd (2012). Comparison of data analysis parameters and MS/MS fragmentation techniques for quantitative proteome analysis using isobaric peptide termini labeling (IPTL). Analytical and Bioanalytical Chemistry. ISSN 1618-2642. 404(4), p. 1103–1114. doi: 10.1007/s00216-012-5949-z. Full text in Research Archive
• Koehler, Christian; Arntzen, Magnus; Strozynski, Margarita Anna; Treumann, Achim & Thiede, Bernd (2011). Isobaric Peptide Termini Labeling Utilizing Site-Specific N-Terminal Succinylation. Analytical Chemistry. ISSN 0003-2700. 83(12), p. 4775–4781. doi: 10.1021/ac200229w. Full text in Research Archive
• Schmidt, Frank; Dahlmann, Burkhardt; Hustoft, Hanne Kolsrud; Koehler, Christian; Strozynski, Margarita Anna & Kloss, Alexander [Show all 9 contributors for this article] (2011). Quantitative proteome analysis of the 20S proteasome of apoptotic Jurkat T cells. Amino Acids. ISSN 0939-4451. 41(2), p. 351–361. doi: 10.1007/s00726-010-0575-6. Full text in Research Archive
• Kozielski, Frank; Riaz, Tahira; DeBonis, Salvatore; Koehler, Christian; Panse, Isabel & Strozynski, Margarita Anna [Show all 9 contributors for this article] (2011). Proteome analysis of microtubule-associated proteins and their interacting partners from mammalian brain. Amino Acids. ISSN 0939-4451. 41(2), p. 363–385. doi: 10.1007/s00726-010-0649-5. Full text in Research Archive
• Arntzen, Magnus; Koehler, Christian; Treumann, Achim & Thiede, Bernd (2011). Quantitative proteome analysis using isobaric peptide termini labeling (IPTL). Methods in molecular biology. ISSN 1064-3745. 753, p. 65–76. doi: 10.1007/978-1-61779-148-2_5. Full text in Research Archive
• Arntzen, Magnus; Koehler, Christian; Barsnes, Harald; Berven, Frode; Treumann, Achim & Thiede, Bernd (2011). IsobariQ: Software for Isobaric Quantitative Proteomics using IPTL, iTRAQ, and TMT. Journal of Proteome Research. ISSN 1535-3893. 10(2), p. 913–920. doi: 10.1021/pr1009977. Full text in Research Archive
• Schmidt, Frank; Dahlmann, Burkhardt; Hustoft, Hanne Kolsrud; Koehler, Christian; Strozynski, Margarita Anna & Klof, Alexander [Show all 9 contributors for this article] (2010). Quantitative proteome analysis of the 20S proteasome of apoptotic Jurkat T cells. Amino Acids. ISSN 0939-4451. doi: 10.1007/s00726-010-0575-6. Full text in Research Archive
• Souza, Gustavo De; Fortuin, Suereta; Aguilar, Diana; Pando, Rogelio Hernandez; McEvoy, Christopher R.E. & Helden, Paul D. van [Show all 10 contributors for this article] (2010). Using a Label-free Proteomics Method to Identify Differentially Abundant Proteins in Closely Related Hypo- and Hypervirulent Clinical Mycobacterium tuberculosis Beijing Isolates. Molecular & Cellular Proteomics. ISSN 1535-9476. 9(11), p. 2414–2423. doi: 10.1074/mcp.M900422-MCP200. Full text in Research Archive Show summary

  While the genome of the M. tuberculosis H37Rv lab strain has been available for over 10 years, it is only recently that genomic information from clinical isolates has been used to generate the hypothesis of virulence differences between different strains. In addition, the relationship between strains displaying differing virulence in an epidemiological setting and their behavior in animal models has received little attention. The potential causes for variation in virulence between strains, as determined by differential protein expression, have similarly been a neglected area of investigation. In this study, we used a label-free quantitative proteomic approach to estimate differences in protein abundance between two closely related Beijing genotypes that have been shown to be hyper- and hypo-virulent on the basis of both epidemiological and mouse model studies. We were able to identify a total of 1670 proteins from both samples, and protein abundance calculations revealed that 48 proteins were over-represented in the hypo-virulent isolate, while 53 were over-represented in the hyper-virulent. Functional classification of these results shows that molecules of cell wall organization and DNA transcription regulatory proteins may have a critical influence in defining the level of virulence. The reduction in the presence of ESAT-6, other Esx-like proteins, and fbpD (MPT51) in the hyper-virulent strain indicates that changes in the repertoire of highly immunogenic proteins can be a defensive process undertaken by the virulent cell. In addition, most of the previously well characterized gene targets related to virulence were found to be similarly expressed in our model. Our data supports the use of proteomics as a complementary tool for genomic comparisons in order to understand the biology of M. tuberculosis virulence.

• Kozielski, Frank; Riaz, Tahira; DeBonis, Salvatore; Koehler, Christian; Kröning, Mario & Panse, Isabel [Show all 9 contributors for this article] (2010). Proteome analysis of microtubule-associated proteins and their interacting partners from mammalian brain. Amino Acids. ISSN 0939-4451. doi: 10.1007/s00726-010-0649-5. Full text in Research Archive
• Dørum, Siri; Arntzen, Magnus; Qiao, Shuo Wang; Holm, Anders; Koehler, Christian & Thiede, Bernd [Show all 8 contributors for this article] (2010). The Preferred Substrates for Transglutaminase 2 in a Complex Wheat Gluten Digest Are Peptide Fragments Harboring Celiac Disease T-Cell Epitopes. PLOS ONE. 5(11). doi: 10.1371/journal.pone.0014056. Full text in Research Archive Show summary

  Conclusion: TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease.

• Tomazella, Gisele Guiqardi; daSilva, Idalete; Thomé, Carolina H.; Greene, Lewis J; Koehler, Christian J & Thiede, Bernd [Show all 8 contributors for this article] (2010). Analysis of detergent-insoluble and whole cell lysate fractions of resting neutrophils using high-resolution mass spectrometry. Journal of Proteome Research. ISSN 1535-3893. 9(4), p. 2030–2036. doi: 10.1021/pr1000253. Full text in Research Archive Show summary

  Neutrophilic granulocytes play a major role in the initiation and resolution of the inflammatory response, and demonstrate significant transcriptional and translational activity. Although much was known about neutrophils prior to the introduction of proteomics, the use of MS-based methodologies has provided an unprecedented tool to confirm and extend previous findings. In the present study, we performed a Gel-LC-MS/MS analysis of neutrophil detergent insoluble and whole cell lysate fractions of resting neutrophils. We achieved a set of identifications through the use of high-resolution mass spectrometry and validation of its data. We identified a total of 1249 proteins with a wide range of intensities from both detergent-insoluble and whole cell lysate fractions, allowing a mapping of proteins such as those involved in intracellular transport (Rab and Sec family proteins) and cell signaling (S100 proteins). These results represent the most comprehensive proteomic characterization of resting human neutrophils to date, and provide important information relevant for further studies of the immune system in health and disease. The methods applied here can be employed to help us understand how neutrophils respond to various physiologic and pathophysiologic conditions and could be extended to protein quantitation after cell activation.

• Saunders, Threrese Solstad; Bjørgo, Elisa; Koehler, Christian; Strozynski, Margarita Anna; Torgersen, Knut Martin & Tasken, Kjetil [Show all 7 contributors for this article] (2010). Quantitative proteome analysis of detergent-resistant membranes identifies the differential regulation of protein kinase C isoforms in apoptotic T cells. Proteomics. ISSN 1615-9853. 10(15), p. 2758–2768. doi: 10.1002/pmic.201000164. Full text in Research Archive
• Koehler, Christian; Strozynski, Margarita Anna; Kozielski, F; Treumann, A & Thiede, Bernd (2009). Isobaric Peptide Termini Labeling for MS/MS-Based Quantitative Proteomics. Journal of Proteome Research. ISSN 1535-3893. 8(9), p. 4333–4341. doi: 10.1021/pr900425n. Full text in Research Archive
• Souza, GA de; Souza, Gustavo De; Softeland, T; Søfteland, Tina; Koehler, Christian J & Thiede, Bernd [Show all 7 contributors for this article] (2009). Validating divergent ORF annotation of the Mycobacterium leprae genome through a full translation dataset and peptide identification by tandem mass spectrometry. Proteomics. ISSN 1615-9853. 9(12), p. 3233–3243. doi: 10.1002/pmic.200800955. Full text in Research Archive Show summary

  Mycobacterium leprae has undergone extensive degenerative evolution, with a large number of pseudogenes. It is also the organism with the greatest divergence between gene annotations from independent institutes. Therefore, M. leprae is a good model to verify the currently predicted coding sequence regions between different annotations, to identify new ones and to investigate the expression of pseudogenes. We submitted a total extract of the bacteria isolated from Armadillo to Gel-LC-MS/MS using a LTQ-Orbitrap mass spectrometer. Spectra were analyzed using the Leproma (1614 genes and 1133 pseudogenes) and TIGR (5446 genes) databases and a database containing the full genome translation. We identified a total of 1046 proteins, including 5 proteins encoded by previously predicted pseudogenes, which upon closer inspection appeared to be proper genes. Only 11 of the additional annotations by TIGR were verified. We also identified 6 tryptic peptides from 5 proteins from regions not considered to be coding sequences, plus peptides from 2 unannotated gene candidates that overlap with other genes. Our data show that the Leproma annotation of M. leprae is quite accurate, and there were no peptide observations corresponding to true pseudogenes, except for a new gene candidate, overlapping with an essential enolase on the complementary strand.

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• 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. Full text in Research Archive
• Thiede, Bernd; Koehler, Christian; Strozynski, Margarita Anna; Treumann, A; Stein, Robert & Zimny-Amdt, Ursula [Show all 8 contributors for this article] (2012). Protein species high resolution quantitative proteomics of HeLa cells using SILAC-2-DE-nanoLC/LTQ-Orbitrap mass spectrometry. Molecular & Cellular Proteomics. ISSN 1535-9476. doi: 10.1074/mcp.M112.019372. Full text in Research Archive
• Arntzen, Magnus; Koehler, Christian; Barsnes, Harald; Berven, Frode; Treumann, Achim & Thiede, Bernd (2011). IsobariQ: Software for isobaric quantitative proteomics using IPTL, iTRAQ and TMT. Full text in Research Archive
• Souza, Gustavo De; Fortuin, Suereta; McEvoy, Christopher R; Pando, Rogelio Hernandez; Koehler, Christian J & Thiede, Bernd [Show all 8 contributors for this article] (2009). A label-free method identified differentially abundant proteins in related M.tuberculosis Beijing strains. Full text in Research Archive

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