Léon Reubsaet

• Mrsa, Ago; Larsen, Bjarke Strøm; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2026). Dual Functionality in Whole Blood Microsampling: Combining Plasma Separation and Immunoaffinity Cleanup for LC-MS Based Targeted Protein Analysis. Journal of Proteome Research. ISSN 1535-3893. 25(9), p. 4665–4675. doi: 10.1021/acs.jproteome.6c00092. Full text in Research Archive
• Halvorsen, Trine Grønhaug; Johannsen, Christina & Reubsaet, Leon (2026). Microsampling of proteins from biological matrices: sampling methods and devices, typical workflows, clinical examples, novel applications and innovations. Analytical Methods. ISSN 1759-9660. doi: 10.1039/d6ay00944a. Full text in Research Archive
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2025). Progress in Dried Microsampling for MS-Based Protein Analysis. Journal of Separation Science. ISSN 1615-9306. 48(7). doi: 10.1002/jssc.70222. Full text in Research Archive
• Mrsa, Ago; Matijevic, Marijana; Dehnes, Yvette; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2025). Comparative Evaluation of Smart Sampling for hCG Determination: A New Potential Direction in Protein Biomarker Analysis From Dried Microsamples. Drug Testing and Analysis. ISSN 1942-7603. 17(12), p. 2390–2398. doi: 10.1002/dta.3948. Full text in Research Archive Show summary

  ABSTRACT Since the early 20th century, sampling biological matrices like blood on paper (dried blood spots [DBS]) has been vital in clinical analysis. While DBS microsampling is convenient for small molecules, extensive sample preparation can make LC‐MS protein analysis impractical because of the time‐consuming steps, especially for low‐abundance proteins. Smart sampling, introduced in 2018, simplifies this by integrating sample preparation directly on the sampler. The work presented in this paper aims to compare a newly validated smart sampling method with two other methods: an in‐house method based on immunocapture on magnetic beads and a commercial method that uses electrochemiluminescence immunoassay (ECLIA). The performance of the three hCG methods was compared using 21 single‐blind serum samples. Linear regression analysis revealed strong correlations (all R 2  > 0.91) between the actual sample concentrations and the results obtained from all three methods. Immunocapture with magnetic beads showed the strongest linear correlation ( R 2  = 0.974). To assess agreement between the methods, Bland–Altman analysis was conducted. The comparison between smart sampling and magnetic beads showed an average bias of −5.2, with no significant trend in variation across the sample concentration range of 0.5–75 ng/mL. The smart sampling and ECLIA comparison revealed a bias of 0.4 ± 4 ng/mL, indicating even better agreement and consistent results. This paper presents the first‐ever comparison of a smart sampling method with existing methods. The results highlight smart sampling as a promising new approach for bioanalysis and boost the technique as a viable alternative in protein biomarker analysis from complex matrices using LC‐MS.

• Johannsen, Christina; Nguyen, Anna Kha Tu; Shabani, Sasha; Oulie, Inger; Dørum, Siri & Dehnes, Yvette [Show all 8 contributors for this article] (2025). Development of smart samplers and their comparison to dried serum spots for the analysis of growth hormone releasing hormone analogs using LC[sbnd]HRMS/MS. Advances in Sample Preparation. 15. doi: 10.1016/j.sampre.2025.100190. Full text in Research Archive
• Mrsa, Ago; Ishaq, Ridja; Brandoli, Laila; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2025). Transitioning from concept to application: Comprehensive analytical validation of antibody-based smart sampling. Journal of Pharmaceutical and Biomedical Analysis. ISSN 0731-7085. 260. doi: 10.1016/j.jpba.2025.116825. Full text in Research Archive
• Al-Rubaye, Noor; Mrsa, Ago; Ramirez, Manuel; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2025). Smart proteolytic samplers for liquid chromatography-mass spectrometry protein quantification: Assessing trypsin immobilization via divinyl sulfone or periodate functionalization. Journal of Chromatography A. ISSN 0021-9673. 1747. doi: 10.1016/j.chroma.2025.465825. Full text in Research Archive Show summary

  In this report we try to answer the question if smart proteolytic samplers can be used for LC-MS based quantitative determination of proteins from paper. Production of smart proteolytic samplers is a two-stage process: In the first stage cellulose is functionalized while in the second stage trypsin is covalently bound. Two functionalization strategies, divinyl sulfone - and periodate functionalization, were compared under various conditions. The divinyl sulfone functionalization in combination with trypsin immobilization is here described for the first time. This functionalization chemistry shows to be equally successful as the periodate functionalization. Using Whatman Grade CF12 paper, it appears that smart proteolytic samplers with high tryptic activity can be produced. These have the capability to produce tryptic peptides with good linearity in buffered solutions: up to 0.998 for BSA (using HLVDEPQNLIK as signature peptide and IO4-functionalization) and 0.990 for Cytochrome C (using MIFAGIK as signature peptide and IO4-functionalization). The reproducibility in complex biological samples is acceptable (<20 % average RSD for divinyl sulfone functionalized paper) when internal correction is carried out. Overall, smart proteolytic samplers can be used for reliable LC-MS based determination of proteins when the correct proteotypic peptides are chosen.

• 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. Full text in Research Archive Show summary

  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.

• Johannsen, Christina; Reubsaet, Leon & Halvorsen, Trine Grønhaug (2024). Revolutionizing biomarker analysis one tip at a time—Coupling antibody to Volumetric Absorptive Microsamplers for sensitive LC-MS analysis. Advances in Sample Preparation. 11. doi: 10.1016/j.sampre.2024.100121. Full text in Research Archive
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2024). Advancements in clinical approaches, analytical methods, and smart sampling for LC–MS-based protein determination from dried matrix spots. Journal of Separation Science. ISSN 1615-9306. 47(9-10). doi: 10.1002/jssc.202400061. Full text in Research Archive
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2023). Is this the end of dried blood spots as we know it? Analytical Science Advances (ASA). 4(7-8), p. 267–274. doi: 10.1002/ansa.202300006. Full text in Research Archive Show summary

  Abstract In 2017 integrated sampling and sample preparation for simplified liquid chromatography‐mass spectrometry analysis of proteins from dried blood spots were introduced. The concept, called smart samplers or smart sampling, enables proteolysis or affinity clean‐up, two common sample preparation steps in liquid chromatography‐mass spectrometric bioanalysis of proteins, to start at the moment of sampling. The idea is to utilize the time for sampling and drying to perform these time‐consuming and labour‐intensive steps. Hence, only a simplified sample preparation is necessary after the arrival of the sample in the lab. In this perspective, we present an overview of the smart sampling approach where the conventional protein analysis workflow is reshuffled to start already prior to arrival in the lab. In addition, we present a thorough discussion of integrating sample preparation steps such as proteolysis or affinity capture in the sampling. Finally, in the end, we try to answer the question if conventional dried blood spots will become obsolete in the future.

• Johannsen, Christina; Mrsa, Ago; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2023). Smart sampling as the “Spot-on” Method for LC-MS protein analysis from dried blood spots. Journal of Separation Science. ISSN 1615-9306. 46(18). doi: 10.1002/jssc.202300394. Full text in Research Archive Show summary

  This perspective explores the feasibility of smart sampling with dried blood spots for the determination of proteins and peptides from human biomatrices using liquid chromatography coupled to mass spectrometry for clinical purposes. The focus is on innovative approaches to transform filter paper from a mere sample carrier to an active element in sample preparation, with the aim of reducing the need for extensive and intensive sample preparation in the conventional sense. Specifically, we discuss the use of modified cellulose to integrate sample preparation at an early stage of sample handling. The use of paper immobilized with either trypsin or monoclonal antibodies for protein digestion and affinity clean‐up is discussed as a potential benefit of starting sample preparation instantly at the moment of sampling to optimize time efficiency and enable faster analysis, diagnosis, and follow‐up of patients.

• Skogvold, Hanne Bendiksen; Rootwelt, Helge; Reubsaet, Leon; Elgstøen, Katja B. Prestø & Wilson, Steven Ray Haakon (2023). Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry. Journal of Separation Science. ISSN 1615-9306. 46(15). doi: 10.1002/jssc.202300210. Full text in Research Archive Show summary

  Dried blood spot samples are simple to prepare and transport, enabling safe andaccessible diagnostics, both locally and globally. We review dried blood spot sam-ples for clinical analysis, focusing on liquid chromatography-mass spectrometryas a versatile measurement tool for these samples. Dried blood spot samplescan provide information for, for example, metabolomics, xenobiotic analysis,and proteomics. Targeted analyses of small molecules are the main applica-tion of dried blood spot samples and liquid chromatography-mass spectrometry,but emerging applications include untargeted metabolomics and proteomics.Applications are highly varied, including analyses related to newborn screen-ing, diagnostics and monitoring of disease progression and treatment effects ofvirtually any disease, as well as studies into the physiology and effects of diet,exercise, xenobiotics, and doping. A range of dried blood spot products andmethods are available, and applied liquid chromatography-mass spectrometryinstrumentation is varied with regard to liquid chromatography column formatsand selectivity. In addition, novel approaches such as on-paper sample prepara-tion (e.g., selective trapping of analytes with paper-immobilized antibodies) aredescribed. We focus on research papers published in the last 5 years.

• Ngo, Huan Bao; Oulie, Inger; Reubsaet, Leon & Halvorsen, Trine Grønhaug (2023). Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins. Journal of Visualized Experiments. 2023(191). doi: 10.3791/64473. Full text in Research Archive
• 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. Full text in Research Archive Show summary

  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.

• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2022). The utility of molecularly imprinted polymers for mass spectrometric protein and proteomics analysis. Proteomics. ISSN 1615-9853. 22. doi: 10.1002/pmic.202100395. Full text in Research Archive Show summary

  Selective and efficient sample clean-up is important in mass spectrometric protein- and proteomics analyses from biological matrices. Molecularly imprinted polymers (MIPs), polymers prepared to have tailor-made cavities for capture of target analytes may by such represent an interesting alternative for selective clean-up. The present review aims to give an overview of the utility of MIPs for protein capture from biological matrices prior to mass spectrometry (MS) analysis. The application of MIPs in depletion of abundant proteins, in protein and proteotypic peptide capture as well as in capture of post-translational modifications (PTMs) is described and discussed. In addition, an overview of available MIP formats and their advantages and challenges is given, together with an overview of the mass spectrometric techniques used in protein analysis after MIP capture. Overall, the present literature demonstrates that for many applications MIPs for sample clean-up in mass spectrometric protein and proteomics analysis from biological matrices is still not fully matured. MIPs for proteotypic peptide capture is the most mature approach and a method for routine use may be available within the next few years.

• 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), p. 171–183. doi: 10.1002/sscp.202100062. Full text in Research Archive Show summary

  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.

• Nielsen, Erik Waage; Miller, Yoav; Brekke, Ole Lars; Grond, Joost; Duong, Anh Hoang & Fure, Hilde [Show all 12 contributors for this article] (2022). A Novel Porcine Model of Ischemia-Reperfusion Injury After Cross-Clamping the Thoracic Aorta Revealed Substantial Cardiopulmonary, Thromboinflammatory and Biochemical Changes Without Effect of C1-Inhibitor Treatment. Frontiers in Immunology. 13:852119, p. 1–12. doi: 10.3389/fimmu.2022.852119. Full text in Research Archive Show summary

  Ischemic injury worsens upon return of blood and innate immunity including the complement system play a central role in ischemia-reperfusion injury (IRI) as in thoracic aortic surgery. Complement component1 inhibitor (C1-INH) has been shown to reduce IRI and is a broad-acting plasma cascade inhibitor. We established a new porcine model of IRI by cross-clamping the thoracic aorta and evaluated the global changes occurring in organ function, systemic inflammatory response and organ damage with or without treatment with C1-INH-concentrate. Twenty-four piglets (8.8-11.1 kg) underwent 45 minutes clamping of the thoracic aorta at the Th8 level. Upfront 12 piglets received human saline and 12 received C1-INH (250 IU/kg) intravenously. Three sham animals received thoracic opening without clamping. Reperfusion lasted 5 hours. We studied ten cardiorespiratory markers, three hematologic markers, eleven inflammatory markers, and twelve organ damage markers over the whole experimental period. Postmortem tissue homogenates from seven organs were examined for inflammatory markers and analysed by two-way repeated-measures ANOVA, area under the curve or unpaired t-tests. By excluding sham and combining treated and untreated animals, the markers reflected a uniform, broad and severe organ dysfunction. The mean and range fold change from before cross-clamp onset to maximum change for the different groups of markers were: cardiorespiratory 1.4 (0.2-3.7), hematologic 1.9 (1.2-2.7), plasma inflammatory 19.5 (1.4-176) and plasma organ damage 2.9 (1.1-8.6). Treatment with C1-INH had only a marginal effect on the IRI-induced changes, reaching statistical significance only for the plasma complement activation product TCC (p=0.0083) and IL-4 (p=0.022) and INF-α (p=0.016) in the colon tissue. In conclusion, the present novel model of porcine global IRI is forceful with regards to central markers and could generally be applicable for pathophysiological studies. C1-INH treatment had no significant effect, but the model allows for future testing of other drugs attenuating IRI globally.

• Johannsen, Christina; Haq, Anam Ul; Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). On the spot immunocapture in targeted biomarker analysis using paper-bound streptavidin as anchor for biotinylated antibodies. Analytical and Bioanalytical Chemistry. ISSN 1618-2642. 414(19), p. 5979–5989. doi: 10.1007/s00216-022-04161-w. Full text in Research Archive Show summary

  Abstract The modification of an easily available resource like paper to circumvent expensive or intensive sample pretreatment could be the answer to sample analysis in resource-poor regions. Therefore, a novel on-paper device combining sample collection with affinity sample pretreatment is introduced here. Universal smart affinity samplers are produced by a simple KIO 4 -mediated oxidation of cellulose, which functionalizes the paper. This is followed by immobilization of streptavidin. Streptavidin serves as a universal anchor for biotinylated antibodies, enabling simple preparation of tailor-made affinity samplers. The functionality of the device was tested using a model protein (human chorionic gonadotropin, hCG) and biotinylated anti-hCG antibodies for affinity capture. In a laboratory setting, the performance was demonstrated, and a 14-fold increase of target binding compared to binding without bmAb was achieved. The recovery of hCG captured with bmAb-treated samplers was determined to be 33% and comparable to previously described affinity capture approaches. Application of the smart affinity samplers to human serum containing hCG showed an R 2 of 0.98 (200–1000 pg mL −1 ), precision of ≤ 9.1% RSD, and estimated limit of detection of 65 pg mL −1 . Although further optimization and validation are necessary prior to application to real samples in clinical settings, the potential of the device for use in determination of low abundant biomarkers in complex samples has been demonstrated.

• 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.

• McKitterick, Nicholas; Bicak, Tugrul Cem; Switnicka-Plak, Magdalena; Cormack, Peter A. G.; Reubsaet, Leon & Halvorsen, Trine Grønhaug (2021). On-line duplex molecularly imprinted solid-phase extraction for analysis of low-abundant biomarkers in human serum by liquid chromatography-tandem mass spectrometry. Journal of Chromatography A. ISSN 0021-9673. 1655. doi: 10.1016/j.chroma.2021.462490. Full text in Research Archive Show summary

  In the present work, a pair of molecularly imprinted polymers (MIPs) targeting distinct peptide targets were packed into trap columns and combined for automated duplex analysis of two low abundant small cell lung cancer biomarkers (neuron-specific enolase [NSE] and progastrin-releasing peptide [ProGRP]). Optimization of the on-line molecularly imprinted solid-phase extraction (MISPE) protocol ensured that the MIPs had the necessary affinity and selectivity towards their respective signature peptide targets – NLLGLIEAK (ProGRP) and ELPLYR (NSE) – in serum. Two duplex formats were evaluated: a physical mixture of the two MIPs (1:1 w/w ratio) inside a single trap column, and two separate MIP trap columns connected in series. Both duplex formats enabled the extraction of the peptides from serum. However, the trap columns in series gave superior extraction efficiency (85.8±3.8% and 49.1±6.7% for NLLGLIEAK and ELPLYR, respectively). The optimized protocol showed satisfactory intraday (RSD≤23.4 %) and interday (RSD≤14.6%) precision. Duplex analysis of NSE and ProGRP spiked into digested human serum was linear (R2≥0.98) over the disease range (0.3-30 nM). The estimated limit of detection (LOD) and limit of quantification (LOQ) were 0.11 nM and 0.37 nM, respectively, for NSE, and 0.06 nM and 0.2 nM, respectively, for ProGRP. Both biomarkers were determined at clinically relevant levels. To the best of our knowledge, the present work is the first report of an automated MIP duplex biomarker analysis. It represents a proof of concept for clinically viable duplex analysis of low abundant biomarkers present in human serum or other biofluids.

• Halvorsen, Trine Grønhaug; McKitterick, Nicholas; Kish, Monika & Reubsaet, Leon (2021). Affinity capture in bottom-up protein analysis – overview of current status of proteolytic peptide capture using antibodies and molecularly imprinted polymers. Analytica Chimica Acta. ISSN 0003-2670. 1182. doi: 10.1016/j.aca.2021.338714. Full text in Research Archive Show summary

  Antibody-based affinity capture has become the gold standard in sample preparation for determination of low-abundance protein biomarkers in biological matrices prior to liquid chromatography-mass spectrometry (LC-MS) determination. This comprises both capture of intact proteins prior to the digestion step and capture of proteolytic peptides after digestion of the sample. The latter can be performed both using antibodies specifically developed to capture target proteolytic peptides, as well as by the less explored use of anti-protein antibodies to capture the proteolytic epitope peptide. Molecularly imprinted polymers (MIPs), also called plastic antibodies are another affinity-based approach emerging as sample preparation technique in LC-MS based protein biomarker analysis. The current review gives a critical and comprehensive overview of proteolytic peptide capture using antibodies and MIPs in LC-MS based protein biomarker determination during the last five years. The main emphasis is on capture of non-modified peptides, while a brief overview of affinity capture of peptides containing post-translational modifications (PTMs) is provided.

• Reubsaet, Leon; Sweredoski, Michael J.; Moradian, Annie; Lomenick, Brett; Eggleston-Rangel, Roxana & Garbis, Spiros D. (2020). Nano volume fractionation strategy for dilute-and-shoot injections in off-line loss-less proteomic workflows for extensive protein identifications of ultra-low sample amounts. Journal of Chromatography A. ISSN 0021-9673. 1609. doi: 10.1016/j.chroma.2019.460507. Full text in Research Archive Show summary

  A proteomic workflow for a simple loss-less manual nano-fractionation (300 nL/fraction) for low µg sample amounts which avoids the need to dry down or transfer fractions to autosampler vials is shown to be feasible. It is demonstrated that the conventional procedure of drying samples down followed by reconstitution negatively affects the number of protein and peptide identifications. Furthermore, these losses seem to disproportionately affect hydrophobic peptides from the drying down and reconstitution step. By collecting and concatenating the fractions while the outlet of the column is submerged in a small predefined volume of 0.2% formic acid, the content of acetonitrile in the collecting vials was lowered such that it was compatible with direct injection for the online analysis. This additionally resulted in a time gain of approx. an hour for the total fractionation time. Acetonitrile concentrations up to 7.5% do not seem to compromise the chromatographic performance in the online analysis. Using as little as 2 µg digested HeLa lysate, approx. 7000 protein groups could be easily identified with 2 or more unique peptides. This was the case when fractionation was performed at pH 10 as well as at pH 5.5.

• McKitterick, Nicholas; Bicak, Tugrul Cem; Cormack, Peter A. G.; Reubsaet, Leon & Halvorsen, Trine Grønhaug (2020). Facilitating serum determination of neuron specific enolase at clinically relevant levels by coupling on-line molecularly imprinted solid-phase extraction to LC-MS/MS. Analytica Chimica Acta. ISSN 0003-2670. 1140, p. 210–218. doi: 10.1016/j.aca.2020.10.022. Full text in Research Archive
• McKitterick, Nicholas; Braathen, Frida; Switnicka-Plak, Magdalena; Cormack, Peter A. G.; Reubsaet, Leon & Halvorsen, Trine Grønhaug (2020). Magnetic Synthetic Receptors for Selective Clean-Up in Protein Biomarker Quantification. Journal of Proteome Research. ISSN 1535-3893. 19(8), p. 3573–3582. doi: 10.1021/acs.jproteome.0c00258. Full text in Research Archive
• Halvorsen, Trine Grønhaug; Rosting, Cecilie; Skjærvø, Øystein & Reubsaet, Leon (2020). Dried Blood Spots in Mass Spectrometry-based Protein Analysis. Recent Developments including Sampling of other Biological Matrices and Novel Sampling Technologies. LC GC Europe. ISSN 1471-6577. 33(5), p. 222–235. Full text in Research Archive Show summary

  The dried blood spot (DBS) sampling technique has been around for decades, predominantly for small molecules and mainly in newborn screening. Although determination of proteins after DBS sampling is usually performed with immunometric assays, the combination with mass spectrometry (MS) is gaining interest. This article provides an overview of DBS sampling for mass spectrometry-based protein analysis. The first part will focus on clinical applications for DBSs and on sampling other biological matrices apart from whole blood, including dried matrix spots (DMSs). The second part will explore the new frontiers of the DBS sampling technology, including novel sampling materials/devices, and novel combinations with mass spectrometry. Examples of use in both qualitative and quantitative protein analysis are highlighted as well as examples using both bottom-up and top-down proteomics approaches.

• Levernæs, Maren Christin Stillesby; Moe, Areli; Bøe, Sigurd; Paus, Elisabeth; Reubsaet, Leon & Halvorsen, Trine Grønhaug (2020). Liquid chromatography mass spectrometry based characterization of epitope configurations. Analytical Methods. ISSN 1759-9660. 12(45), p. 5476–5484. doi: 10.1039/d0ay01283a. Full text in Research Archive
• Levernæs, Maren Christin Stillesby; Farhat, Bassem; Oulie, Inger; Abdullah, Sazan Salih; Paus, Elisabeth & Reubsaet, Leon [Show all 7 contributors for this article] (2019). Immunocapture sample clean-up in determination of low abundant protein biomarkers-a feasibility study of peptide capture by anti-protein antibodies. RSC Advances. 9(60), p. 34902–34911. doi: 10.1039/c9ra05071j. Full text in Research Archive Show summary

  Immunocapture in mass spectrometry based targeted protein analysis using a bottom-up workflow is nowadays mainly performed by target protein extraction using anti-protein antibodies followed by tryptic digestion. Already available monoclonal antibodies (mAbs) which were developed against intact target proteins (anti-protein antibodies) can capture proteotypic epitope containing peptides after tryptic digestion of the sample. In the present paper considerations when developing a method for targeted protein quantitation through capture of epitope containing peptides are discussed and a method applying peptide capture by anti-protein antibodies is compared with conventional immunocapture MS. The model protein used for this purpose was progastrin releasing peptide (ProGRP), a validated low abundant biomarker for Small Cell Lung Cancer with reference values in serum in the pg mL−1 range. A set of mAbs which bind linear epitopes of ProGRP are available, and after a theoretical consideration, three mAbs (E146, E149 and M18) were evaluated for extraction of proteotypic epitope peptides from a complex sample. M18 was the best performing mAb for peptide capture by anti-protein antibodies, matching the LOD (54 pg mL−1) and LOQ (181 pg mL−1) of the existing conventional immunocapture LC-MS/MS method for determination of ProGRP. Peptide and protein capture using the same mAb were also compared with respect to sample clean-up, and the peptide capture workflow yielded cleaner extracts and therewith less complex chromatograms. Analysis of five patient samples demonstrated that peptide capture by anti-protein antibodies can be used for the determination of various levels of endogenously present ProGRP.

• Skjærvø, Øystein; Solbakk, Eirik Johan Winther; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2019). Paper-based immunocapture for targeted protein analysis. Talanta: The International Journal of Pure and Applied Analytical Chemistry. ISSN 0039-9140. 195, p. 764–770. doi: 10.1016/j.talanta.2018.12.013. Full text in Research Archive
• Skjærvø, Øystein; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2019). All-in-one paper-based sampling chip for targeted protein analysis. Analytica Chimica Acta. ISSN 0003-2670. 1089, p. 56–65. doi: 10.1016/j.aca.2019.08.043. Full text in Research Archive
• Skjærvø, Øystein; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2019). Pre-lab proteolysis for dried serum spots – a paper-based sampling concept targeting low abundant biomarkers. Analytical Methods. ISSN 1759-9660. 12(1), p. 97–103. doi: 10.1039/c9ay01976f. Full text in Research Archive
• Reubsaet, Leon; Sweredoski, Michael J. & Moradian, Annie (2019). Data-Independent Acquisition for the Orbitrap Q Exactive HF: A Tutorial. Journal of Proteome Research. ISSN 1535-3893. 18(3), p. 803–813. doi: 10.1021/acs.jproteome.8b00845. Full text in Research Archive Show summary

  Data-independent acquisition (DIA) is a powerful mass spectrometric technique to perform both protein identification and quantification of complex protein samples. Setting up DIA methods on Orbitrap analyzers requires a thorough overview of the actions the Orbitrap mass spectrometers carry out. This Tutorial is written with the intention to give an overview of the important parameters to consider as well as which measurements to carry out to get the most out of your DIA method when setting it up on an Orbitrap mass analyzer. Instead of giving the optimal DIA settings, all steps in the construction and optimization of the DIA method are shown and discussed in a way that allows tailored DIA methods. They key steps are building the spectral library after sample fractionation, deciding upon the number of data points per chromatographic peak, determining the scan times of each mass spectrometric step, constructing various DIA methods using these data, and evaluating their performance. This proposed DIA method development strategy was tested on digested lysates from Pseudomonas aeruginosa and compared with conventional DDA analysis to put the DIA results into perspective.

• Skjærvø, Øystein; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2018). Smart blood spots for whole blood protein analysis. The Analyst. ISSN 0003-2654. 143(13), p. 3184–3190. doi: 10.1039/c8an00317c. Full text in Research Archive
• Levernæs, Maren Christin Stillesby; Brandtzaeg, Ole Kristian; Amundsen, Sunniva Furre; Reubsaet, Leon; Lundanes, Elsa & Halvorsen, Trine Grønhaug [Show all 7 contributors for this article] (2018). Selective Fishing for Peptides with Antibody Immobilized Acrylate Monoliths, Coupled Online with NanoLC-MS. Analytical Chemistry. ISSN 0003-2700. 90, p. 13860–13860. doi: 10.1021/acs.analchem.8b00935. Full text in Research Archive
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2017). Antibody based affinity capture LC-MS/MS in quantitative determination of proteins in biological matrices. TrAC. Trends in analytical chemistry. ISSN 0165-9936. 95, p. 132–139. doi: 10.1016/j.trac.2017.08.009. Full text in Research Archive Show summary

  Determination of proteins in complex biological matrices has massive attention and is exploited in many different scientific disciplines. Routinely, proteins are determined using antibody based immuno-metric assays. Although these assays are easy to perform and widely used, interpretation of the results is challenging: cross reactivity, high dose hook-effect, presence of heterophile- or auto-antibodies give rise to false results, sometimes with dramatic consequences. In the quest for more robust assays a combination of antibody sample clean-up, tryptic digestion and mass spectrometric determination is gaining more attention. This review discusses the advantages of antibody based affinity capture and subsequent LC-MS/MS in protein analysis like less false results and possibilities like multiplexing and isoform differentiation. It also considers the interplay between the analytical, biological and biochemical factors, which still give rise to false results, even with mass spectrometry as the ultimate selective detection step. The intention of this review is to point out both strengths and weaknesses of antibody based affinity capture LC-MS/MS in quantitative determination of proteins in biological matrices.

• Rossetti, Cecilia; Ore, Odd Gøran; Sellergren, Börje; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2017). Exploring the peptide retention mechanism in molecularly imprinted polymers. Analytical and Bioanalytical Chemistry. ISSN 1618-2642. 409(24), p. 5631–5643. doi: 10.1007/s00216-017-0520-6. Full text in Research Archive Show summary

  Molecularly imprinted polymers (MIPs) have been used as useful sorbents in solid-phase extraction for a wide range of molecules and sample matrices. Their unique selectivity can be fine-tuned in the imprinting process and is crucial for the extraction of macromolecules from complex matrices such as serum. A relevant example of this is the application of MIPs to peptides in diagnostic assays. In this article the selectivity of MIPs, previously implemented in a quantitative mass-spectrometric assay for the biomarker pro-gastrin-releasing peptide, is investigated. Partial least squares regression was used to generate models for the evaluation and prediction of the retention mechanism of MIPs. A hypothesis on interactions of MIPs with the target peptide was verified by ad hoc experiments considering the relevant peptide physicochemical properties highlighted from the multivariate analysis. Novel insights into and knowledge of the driving forces responsible for the MIP selectivity have been obtained and can be directly used for further optimization of MIP imprinting strategies.

• Jagadeesan, Kishore Kumar; Rossetti, Cecilia; Qader, Abed Abdel; Reubsaet, Leon; Sellergren, Börje & Laurell, Thomas [Show all 7 contributors for this article] (2017). Filter Plate?Based Screening of MIP SPE Materials for Capture of the Biomarker Pro-Gastrin-Releasing Peptide. SLAS Discovery. ISSN 2472-5552. 22(10), p. 1253–1261. doi: 10.1177/2472555216689494. Full text in Research Archive
• Rossetti, Cecilia; Switnicka-Plak, Magdalena; Halvorsen, Trine Grønhaug; Cormack, Peter A G; Sellergren, Börje & Reubsaet, Leon (2017). Automated protein biomarker analysis: On-line extraction of clinical samples by Molecularly Imprinted Polymers. Scientific Reports. 7. doi: 10.1038/srep44298. Full text in Research Archive Show summary

  Robust biomarker quantification is essential for the accurate diagnosis of diseases and is of great value in cancer management. In this paper, an innovative diagnostic platform is presented which provides automated molecularly imprinted solid-phase extraction (MISPE) followed by liquid chromatography-mass spectrometry (LC-MS) for biomarker determination using ProGastrin Releasing Peptide (ProGRP), a highly sensitive biomarker for Small Cell Lung Cancer, as a model. Molecularly imprinted polymer microspheres were synthesized by precipitation polymerization and analytical optimization of the most promising material led to the development of an automated quantification method for ProGRP. The method enabled analysis of patient serum samples with elevated ProGRP levels. Particularly low sample volumes were permitted using the automated extraction within a method which was time-efficient, thereby demonstrating the potential of such a strategy in a clinical setting.

• Levernæs, Maren Christin Stillesby; Nordlund, Marianne Sparby; Reubsaet, Leon & Halvorsen, Trine Grønhaug (2017). To elute or not to elute in immunocapture bottom-up LC?MS. Journal of chromatography. B. ISSN 1570-0232. 1055-1056, p. 51–60. doi: 10.1016/j.jchromb.2017.03.044. Full text in Research Archive Show summary

  Immunocapture-based bottom-up LC–MS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads. Two previously validated immunocapture based MS methods for the quantification of pro-gastrin releasing peptide (ProGRP) and human chorionic gonadotropin (hCG) were used as model systems. The tryptic peptide generation was shown to be protein dependent and influenced by protein folding and accessibility towards trypsin both on-beads and in the eluate. The elution of proteins bound to the beads was also shown to be incomplete. In addition, the on-beads digestion suffered from non-specific binding of the trypsin generated peptides. A combination of on-beads digestion and elution may be applied to improve both the quantitative (peak area of the signature peptides) and qualitative yield (number of missed cleavages, total number of identified peptides, coverage, signal intensity and number of zero missed cleavage peptides) of the target proteins. The quantitative yield of signature peptides was shown to be reproducible in all procedures tested.

• Skjærvø, Øystein; Rosting, Cecilie; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2017). Instant on-paper protein digestion during blood spot sampling. The Analyst. ISSN 0003-2654. 142(20), p. 3837–3847. doi: 10.1039/c7an01075c. Full text in Research Archive Show summary

  A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.

• Rossetti, Cecilia; Levernæs, Maren Christin Stillesby; Reubsaet, Leon & Halvorsen, Trine Grønhaug (2016). Evaluation of affinity-based serum clean-up in mass spectrometric analysis: Plastic vs monoclonal antibodies. Journal of Chromatography A. ISSN 0021-9673. 1471, p. 19–26. doi: 10.1016/j.chroma.2016.09.069. Full text in Research Archive
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2016). Determination of very low-abundance diagnostic proteins in serum using immuno-capture LC-MS-MS. LC GC Europe. ISSN 1471-6577. 29(7). Full text in Research Archive Show summary

  The use of antibodies in “bottom-up “LC-MS workflows to determine low abundant biological active proteins in complex human samples has increased in recent years: immuno-capture analysis combines the workflow of conventional immunological assays with LC–MS analysis. This paper describes typical challenges, such as cross reactivity and the mass spectrometer’s dynamic range, as well as the advantages of isoform differentiation and multiplexing. Additionally, some experimental formats of immuno-capture bottom-up LC–MS analysis of biological active proteins in complex human samples will be discussed.

• Egeland, Siri Valen; Reubsaet, Leon & Halvorsen, Trine Grønhaug (2016). The pros and cons of increased trypsin-to-protein ratio in targeted protein analysis. Journal of Pharmaceutical and Biomedical Analysis. ISSN 0731-7085. 123, p. 155–161. doi: 10.1016/j.jpba.2016.02.011. Full text in Research Archive
• Fagerlund, Annette; Smith, Veronika; Kjendseth, Åsmund Røhr; Lindbäck, Toril; Parmer, Marthe Petrine & Andersson, K. Kristoffer [Show all 8 contributors for this article] (2016). Cyclic diguanylate regulation of Bacillus cereus group biofilm formation. Molecular Microbiology. ISSN 0950-382X. 101(3), p. 471–494. doi: 10.1111/mmi.13405. Full text in Research Archive Show summary

  Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram-negatives, increased levels of the second messenger cyclic diguanylate (c-di-GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c-di-GMP include cell division, differentiation and virulence. Among Gram-positive bacteria, where the function of c-di-GMP signaling is less well characterized, c-di-GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c-di-GMP signaling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c-di-GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c-di-GMP signalling between B. subtilis and B. cereus group bacteria.

• Egeland, Siri Valen; Reubsaet, Leon; Paus, Elisabeth & Halvorsen, Trine Grønhaug (2016). Dual-immuno-MS technique for improved differentiation power in heterodimeric protein biomarker analysis: determination and differentiation of human chorionic gonadotropin variants in serum. Analytical and Bioanalytical Chemistry. ISSN 1618-2642. 408(26), p. 7378–7391. doi: 10.1007/s00216-016-9818-z. Full text in Research Archive
• Hildonen, Siri; Skarpen, Ellen; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2016). Isolation and mass spectrometry analysis of urinary extraexosomal proteins. Scientific Reports. 6. doi: 10.1038/srep36331. Full text in Research Archive Show summary

  The aim of the present study was to develop a LC-MS/MS-based proteomic analysis method of urinary exosomal proteins that has the potential to discover disease biomarkers. In short, urinary exosomes from healthy subjects were isolated by immunocapture on magnetic beads, detected by immunofluorescence and TEM, trypsin digested directly on the beads for an accelerated time with no addition of detergents before performing an LC-MS analysis of the trypsinate. To our knowledge, this is the first proteomic analysis of proteins displayed on the outer surface of exosomes. The outer exosome proteome may contain proteins that are of higher biomarker value compared to soluble cargo protein as the proteins projecting into the extracellular milieu might be more directly involved in physiological functions of exosomes. The proteomic analysis identified 49 proteins that were considered significant; the majority is involved in carbohydrate and lipid metabolism or in immune responses. Thirty of the proteins are linked to diseases. The developed proteomic method exploiting urinary exosomes might be of great value in search for diagnostic or prognostic biomarkers of especially metabolic and immune-related diseases.

• Ringvold, Amund & Reubsaet, Leon (2016). The impact of high-dose acetylcholine on bovine corneal epithelium. Acta Ophthalmologica. ISSN 1755-375X. 94(2), p. 160–164. doi: 10.1111/aos.12889. Full text in Research Archive
• Brudal, Espen; Lampe, Elisabeth Opsjøn; Reubsaet, Leon; Roos, Norbert; Hegna, Ida Kristin & Thrane, Ida Marie [Show all 8 contributors for this article] (2015). Vaccination with outer membrane vesicles from Francisella noatunensis reduces development of francisellosis in a zebrafish model. Fish and Shellfish Immunology. ISSN 1050-4648. 42(1), p. 50–57. doi: 10.1016/j.fsi.2014.10.025. Full text in Research Archive
• Reubsaet, Leon (2015). High-performance liquid chromatography (HPLC) and high-performance liquid chromatography-mass spectrometry (LC-MS). In Hansen, Steen Honoré & Pedersen-Bjergaard, Stig (Ed.), Bioanalysis of pharmaceuticals : sample preparation, separation techniques and mass spectrometry. Wiley-Blackwell. ISSN 9781118716823. p. 123–172. doi: 10.1002/9781118716830.ch7. Full text in Research Archive
• Reubsaet, Leon (2015). Physicochemical properties of drug substances. In Hansen, Steen Honoré & Pedersen-Bjergaard, Stig (Ed.), Bioanalysis of pharmaceuticals : sample preparation, separation techniques and mass spectrometry. Wiley-Blackwell. ISSN 9781118716823. p. 9–22. doi: 10.1002/9781118716830.ch2. Full text in Research Archive
• Rossetti, Cecilia; Qader, Abed Abdel; Halvorsen, Trine Grønhaug; Sellergren, Börje & Reubsaet, Leon (2014). Antibody-free biomarker determination: Exploring molecularly imprinted polymers for pro-gastrin releasing peptide. Analytical Chemistry. ISSN 0003-2700. 86(24), p. 12291–12298. doi: 10.1021/ac503559c. Full text in Research Archive
• Torsetnes, Silje Bøen; Levernæs, Maren Christin Stillesby; Nordlund, Marianne Sparby; Paus, Elisabeth; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2014). Multiplexing determination of small cell lung cancer biomarkers and their isovariants in serum by immunocapture LC-MS/MS. Analytical Chemistry. ISSN 0003-2700. 86(14), p. 6983–6992. doi: 10.1021/ac500986t. Full text in Research Archive
• Qader, Abed Abdel; Urraca, Javier; Torsetnes, Silje Bøen; Tønnesen, Finn; Reubsaet, Leon & Sellergren, Börje (2014). Peptide imprinted receptors for the determination of the small cell lung cancer associated biomarker progastrin releasing peptide. Journal of Chromatography A. ISSN 0021-9673. 1370, p. 56–62. doi: 10.1016/j.chroma.2014.10.023. Full text in Research Archive
• Torsetnes, Silje Bøen; Nordlund, Marianne Sparby; Paus, Elisabeth; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2014). Determining ProGRP and isoforms in lung and thyroid cancer patient samples: Comparing an MS method with a routine clinical immunoassay. Analytical and Bioanalytical Chemistry. ISSN 1618-2642. 406(11), p. 2733–2738. doi: 10.1007/s00216-014-7634-x. Full text in Research Archive
• Lund, Hanne; Paus, Elisabeth; Berger, Peter; Stenman, Ulf-Håkan; Torcellini, Tamara & Halvorsen, Trine Grønhaug [Show all 7 contributors for this article] (2014). Epitope analysis and detection of human chorionic gonadotropin (hCG) variants by monoclonal antibodies and mass spectrometry. Tumour Biology. ISSN 1010-4283. 35(2), p. 1013–1022. doi: 10.1007/s13277-013-1135-y. Full text in Research Archive
• Seip, Knut Fredrik; Bjerknes, Kari Christiane; Johansen, Harald Thidemann; Nielsen, Erik Waage; Landrø, Linn Aina Ysland & Reubsaet, Leon (2014). Bradykinin analysis revived - A validated method for determination of its stable metabolite in whole blood by LC-MS/MS. Journal of chromatography. B. ISSN 1570-0232. 947-948, p. 139–144. doi: 10.1016/j.jchromb.2013.12.033. Full text in Research Archive
• Hildonen, Siri; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2014). Why less is more when generating tryptic peptides in bottom-up proteomics. Proteomics. ISSN 1615-9853. 14(17-18), p. 2031–2041. doi: 10.1002/pmic.201300479. Full text in Research Archive
• Vestrheim, Anne Cathrine; Moen, Anders; Egge-Jacobsen, Wolfgang; Reubsaet, Leon; Halvorsen, Trine Grønhaug & Bratlie, Diane Bryant [Show all 8 contributors for this article] (2014). A pilot study showing differences in glycosylation patterns of IgG subclasses induced by pneumococcal, meningococcal, and two types of influenza vaccines. Immunity,Inflammation and Disease. 2(2), p. 76–91. doi: 10.1002/iid3.22. Full text in Research Archive

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• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2020). 100 years of hCG. Elsevier. ISBN 9780128200506. 331 p. Full text in Research Archive

View all works in NVA

• Reubsaet, Leon & Bergquist, Jonas (2025). Special Issue on the 18^thNordic Mass Spectrometry Meeting in Geilo, Norway. Journal of Mass Spectrometry. ISSN 1076-5174. 60(12). doi: 10.1002/jms.5192. Full text in Research Archive
• Reubsaet, Leon (2024). Smart Sampling: next generation DBS samplers for LC-MS based protein analysis. Full text in Research Archive
• Reubsaet, Leon (2024). Smart Sampling: next generation DBS samplers for LC-MS based protein analysis. Full text in Research Archive
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2023). Smart sampling: next-generation DBS samplers for LC-MS based protein analysis. Full text in Research Archive
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2023). Next generation dried blood spot samplers for simplified LC-MS/MS analysis of proteins. Full text in Research Archive
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2023). Smart samplers for simplified LC-MS/MS analysis of proteins. Full text in Research Archive
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2023). Smart samplers for mass spectrometric bioanalysis of proteins. Full text in Research Archive
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2022). Syntetisk antistoff forenklar analyse av blodprøve. [Journal]. Forskning.no. Full text in Research Archive
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2022). Smart samplers in mass spectrometric bioanalysis of proteins. Full text in Research Archive
• Vik, Anders; Halvorsen, Trine Grønhaug; Primdahl, Karoline Gangestad; Hansen, Trond Vidar; Wangensteen, Helle & Reubsaet, Leon [Show all 7 contributors for this article] (2022). Grunnkurs i farmasøytisk kjemi på UiO moderniseres kontinuerlig. Norsk Farmaceutisk Tidsskrift (NFT). ISSN 0029-1935. p. 33–34. Full text in Research Archive
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). Smart Samplers for LC-MS based protein analysis. Full text in Research Archive
• Reubsaet, Leon (2022). Hvordan vedlikeholder jeg LC-MS instrumentet. Full text in Research Archive
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). Smart Samplers for LC-MS based protein analysis. Full text in Research Archive
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). Smart samplers for LC-MS based protein analysis. Full text in Research Archive
• Reubsaet, Leon (2022). Smart Sampler for LC-MS based protein analysis. Full text in Research Archive
• Reubsaet, Leon (2022). Smart Sampler for LC-MS based protein analysis. Full text in Research Archive
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). Smart samplers for LC-MS based protein analysis. Full text in Research Archive
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). Smart Sampler for LC-MS based protein analysis. Full text in Research Archive
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2021). Advances in sampling and sample preparation for mass spectrometric determination of protein biomarkers. Full text in Research Archive
• Bakketeig, Eris Aas; Olsen, Christine; Berg, Henriette Engen; Nyman, Tuula Anneli; Thiede, Bernd & Lundanes, Elsa [Show all 8 contributors for this article] (2021). Optimization of concatenation of fractions in comprehensive two dimensional liquid chromatography bottom-up proteomics. Full text in Research Archive Show summary

  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.

• Reubsaet, Leon (2020). Ekstremt små blodprøvar kan analyserast utan tap av informasjon. [Internet]. Titan.uio.no. Full text in Research Archive
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2019). Affinity based sample clean-up prior to LC-MS/MS for determination of low abundance proteins. Full text in Research Archive
• Skjærvø, Øystein; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2019). Smart blood-spot sampling for MS based bottom-up protein analysis. Full text in Research Archive
• Reubsaet, Leon (2019). Forsker på ny teknologi for diagnostikk i Los Angeles. [Journal]. Dagens Medisin. Full text in Research Archive
• Skjærvø, Øystein; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2019). Smart blood spot sampling for MS tased bottom-up protein analysis. Full text in Research Archive
• Brandtzaeg, Ole Kristian; Levernæs, Maren Christin Stillesby; Halvorsen, Trine Grønhaug; Reubsaet, Leon; Røen, Bent Tore & Vehus, Tore [Show all 10 contributors for this article] (2018). Online sample processing systems for targeted nano liquid chromatography mass spectrometry bioanalysis. Full text in Research Archive
• Skjærvø, Øystein; Pilarova, Veronika; Khalikova, Maria; Reubsaet, Leon; Halvorsen, Trine Grønhaug & Gjelstad, Astrid [Show all 10 contributors for this article] (2018). Those Who Can, Teach: Pharma Stars. The Analytical Scientist. ISSN 2051-4077. 1118, p. 40–45. Full text in Research Archive
• Skjærvø, Øystein; Pilarova, Veronika; Khalikova, Maria; Reubsaet, Leon; Halvorsen, Trine Grønhaug & Gjelstad, Astrid [Show all 10 contributors for this article] (2018). Those Who Can, Teach: Pharma Stars. The Analytical Scientist. ISSN 2051-4077. 1118, p. 40–45. Full text in Research Archive
• Rossetti, Cecilia; Ore, Odd Gøran; Sellergren, Börje; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2017). Correction to: Exploring the peptide retention mechanism in molecularly imprinted polymers (Analytical and Bioanalytical Chemistry, (2017), 409, 24, (5631-5643), 10.1007/s00216-017-0520-6). Analytical and Bioanalytical Chemistry. ISSN 1618-2642. 410(5), p. 1607–1608. doi: 10.1007/s00216-017-0797-5. Full text in Research Archive
• Halvorsen, Trine Grønhaug; Rossetti, Cecilia; Levernæs, Maren Christin Stillesby & Reubsaet, Leon (2016). The Impact of Sample Clean-up Technique in Bottom-up based LC-MS Analysis of Low Abundance Serum Biomarkers. Full text in Research Archive
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2015). Analysis of Peptide and Protein Drugs in Biological Fluids. In Hansen, Steen Honoré & Pedersen-Bjergaard, Stig (Ed.), Bioanalysis of pharmaceuticals : sample preparation, separation techniques and mass spectrometry. Wiley-Blackwell. ISSN 9781118716823. p. 261–282. Full text in Research Archive
• Brandtzæg, Ole Kristian Merkesvik; Hustoft, Hanne Kolsrud; Vehus, Tore; Greibrokk, Tyge; Krauss, Stefan & Reubsaet, Leon [Show all 8 contributors for this article] (2015). On-line protein digestion and peptide separation using “ultra-nano” LC-MS. Full text in Research Archive
• Reubsaet, Leon (2015). Targeted Proteomics - Immunoaffinity sample preparation and isoform differentiation. Full text in Research Archive
• Halvorsen, Trine Grønhaug; Egeland, Siri Valen & Reubsaet, Leon (2015). Dual immunocapture combined with targeted LC-MS/MS for increased differentiation power in protein biomarker determination. Full text in Research Archive
• Myhrer, Marthe; Krogstad, Veronica; Christensen, Hege Staaland; Reubsaet, Leon & Hermann, Monica (2015). Kvantifisering av P-glykoprotein i lymfocytter ved bruk av LC-MS/MS. Full text in Research Archive
• Halvorsen, Trine Grønhaug; Lund, Hanne & Reubsaet, Leon (2014). Immunoaffinity + LC-MS/MS: An alternative approach for antibody selectivity evaluation? Full text in Research Archive
• Reubsaet, Leon (2014). Bestemmelse av proteiner ved hjelp av LC-MS. Full text in Research Archive
• Hustoft, Hanne Kolsrud; Brandtzæg, Ole Kristian Merkesvik; Torsetnes, Silje Bøen; Greibrokk, Tyge; Reubsaet, Leon & Wilson, Steven Ray Haakon [Show all 7 contributors for this article] (2014). Development of a nanoproteomic platform with integrated immunocapture and enzymatic digestion for on-line detection of the low abundant cancer biomarker PROGRP. Full text in Research Archive
• Bakketeig, Eris Aas; Wilson, Steven Ray Haakon; Lundanes, Elsa; Berg, Henriette Engen; Olsen, Christine & Reubsaet, Leon [Show all 8 contributors for this article] (2021). The setup and optimization of a complete global proteomics two-dimensional liquid chromatography system, using Spider Fractionation. Universitetet i Oslo. Full text in Research Archive
• Brandtzaeg, Ole Kristian; Wilson, Steven Ray Haakon; Lundanes, Elsa; Reubsaet, Leon; Krauss, Stefan & Martinsen, Ørjan Grøttem (2018). Online sample Preparation Platforms for Targeted Nano Liquid Chromatography Mass Spectrometry Bioanalysis. Universitetet i Oslo. Full text in Research Archive
• Hustoft, Hanne Kolsrud; Lundanes, Elsa; Wilson, Steven Ray Haakon; Reubsaet, Leon & Greibrokk, Tyge (2014). Accelerating enzymatic protein digestion and on-line nano liquid chromatography-mass spectrometry based proteomics. Universitetet i Oslo. Full text in Research Archive

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