Trine Grønhaug Halvorsen

• 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), s. 4665–4675. doi: 10.1021/acs.jproteome.6c00092. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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), s. 2390–2398. doi: 10.1002/dta.3948. Fulltekst i vitenarkiv Vis sammendrag

  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 [Vis alle 8 forfattere av denne artikkelen] (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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv Vis sammendrag

  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. 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.

• Rye, Torstein Kige; Lee, Chien-Yun; Zellner, Andreas; Moen, Sara Haglund; Dowlatshah, Samira & Halvorsen, Trine Grønhaug [Vis alle 8 forfattere av denne artikkelen] (2024). Electromembrane extraction of peptides based on charge, hydrophobicity, and size – A large-scale fundamental study of the extraction window. Journal of Separation Science. ISSN 1615-9306. 47(15). doi: 10.1002/jssc.202400292. Fulltekst i vitenarkiv Vis sammendrag

  This study investigated the capability of electromembrane extraction (EME) as a general technique for peptides, by extracting complex pools of peptides comprising in total of 5953 different substances, varying in size from seven to 16 amino acids. Electromembrane extraction was conducted from a sample adjusted to pH 3.0 and utilized a liquid membrane consisting of 2-nitrophenyl octyl ether and carvacrol (1:1 w/w), containing 2% (w/w) di(2-ethylhexyl) phosphate. The acceptor phase was 50 mM phosphoric acid (pH 1.8), the extraction time was 45 min, and 10 V was used. High extraction efficiency, defined as a higher peptide signal in the acceptor than the sample after extraction, was achieved for 3706 different peptides. Extraction efficiencies were predominantly influenced by the hydrophobicity of the peptides and their net charge in the sample. Hydrophobic peptides were extracted with a net charge of +1, while hydrophilic peptides were extracted when the net charge was +2 or higher. A computational model based on machine learning was developed to predict the extractability of peptides based on peptide descriptors, including the grand average of hydropathy index and net charge at pH 3.0 (sample pH). This research shows that EME has general applicability for peptides and represents the first steps toward in silico prediction of extraction efficiency.

• Dowlatshah, Samira; Rye, Torstein Kige; Hansen, Frederik Andre; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2024). Parallel electromembrane extraction of peptides with monoterpene and medium-length fatty acid deep eutectic solvents. Analytica Chimica Acta. ISSN 0003-2670. 1297. doi: 10.1016/j.aca.2024.342360. Fulltekst i vitenarkiv Vis sammendrag

  Background Electromembrane extraction (EME) involves the process of mass transfer of charged analytes from an aqueous sample through an organic liquid membrane into an aqueous acceptor medium under the influence of an electrical field. Successful solvation of the analyte within the liquid membrane is of paramount importance and involves molecular interactions with the liquid membrane. In this comprehensive investigation, parallel EME was examined using a training set of 13 model peptides employing deep eutectic solvents as the liquid membrane. These deep eutectic solvents were formulated by mixing specific monoterpenes (thymol, menthol, camphor) with medium-chain fatty acids (1-octanoic acid and 1-decanoic acid). Results From an array of different liquid membrane compositions explored, it was revealed that the combination of camphor and 1-decanoic acid (in a 1:1 w/w ratio) with 2% di (2-ethylhexyl) phosphate (DEHP) delivered the most efficient extraction system. The solvation of the model peptides within this liquid membrane predominantly relied on ionic interactions between protonated basic functionalities and DEHP, along with hydrogen bond interactions between the deprotonated acid functionalities (hydrogen bond acceptor) and 1-decanoic acid (hydrogen bond donor). Selectivity was modulated by the pH of the sample and acceptor solutions, with a direct correlation to the polarity and net charge of the model peptides. The ionization of 1-decanoic acid in the interfacial region between the sample and liquid membrane emerged as an important factor influencing the selectivity. Significance and novelty Although parallel EME of peptides has been reported previously, the current liquid membrane provides an extraction system with sufficient stability for the first time. Selective extraction of peptides through EME holds substantial promise within the realm of next-generation environmentally-friendly sample preparation methodologies. The findings presented in this paper contribute significantly to our fundamental understanding of these processes, and may serve as an important reference for the development of future methods in this field.

• Rye, Torstein Kige; Dowlatshah, Samira; Hansen, Frederik Andre; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2024). Electromembrane extraction of peptides – Looking retrospectively into published studies. Advances in Sample Preparation. 9. doi: 10.1016/j.sampre.2024.100106. Fulltekst i vitenarkiv Vis sammendrag

  Electromembrane extraction (EME) has been developed into an attractive microextraction concept for extraction of pharmaceuticals and related substances from blood and urine. In EME, charged substances are extracted across a liquid membrane (organic) under the influence of an electrical field. Recently, commercial equipment for EME was launched, and this is expected to increase the interest for the technique. EME has also been explored for extraction of peptides, but this is more complicated due to the challenges related to the solvation of peptides in the organic liquid membrane. In this paper, we review the literature on EME of peptides, and we retrospectively look closer into published studies based on our current understanding of the concept. By such, we can explain early observations in more detail, and we can improve our fundamental understanding.

• Rye, Torstein Kige; Hansen, Frederik Andre; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2024). Electromembrane extraction of human chorionic gonadotropin – A case study on mass transfer of intact protein versus signature peptide. Advances in Sample Preparation. 11. doi: 10.1016/j.sampre.2024.100126. Fulltekst i vitenarkiv Vis sammendrag

  For the first time, we present targeted protein detection by tryptic digestion of human chorionic gonadotropin (hCG) followed by electromembrane extraction (EME). Operational parameters were optimized, and urine and serum samples spiked with hCG underwent tryptic digestion followed by EME of the βT5 signature peptide. The liquid membrane comprised nitrophenyl octyl ether (NPOE), carvacrol, and di(2-ethyl hexyl) phosphate (DEHP) at ratios of 49:49:2 (w/w/w). Extractions were performed in a conductive vial format for 45 min at 5 V. Even from highly complex digested samples of serum and urine, the signature peptide βT5 was extracted by EME and detected by LC-MS/MS. While attempts to extract intact hCG protein were unsuccessful, the extraction of the signature peptide was efficient. The extraction recovery from undigested and digested urine was 71 % (RSD = 17 %) and 116 % (RSD = 17 %), respectively. For serum, the extraction recoveries were 11 % (RSD = 23 %) for undigested samples and 110 % (RSD = 14 %) for digested samples. This study demonstrates both the potential and challenges of EME for protein analysis. Experiments regarding EME of intact proteins provided new insights into protein phase distribution. This fundamental case study underscores the potential of EME as a sample preparation technique for the targeted determination of protein biomarkers and drugs.

• 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. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• 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), s. 267–274. doi: 10.1002/ansa.202300006. Fulltekst i vitenarkiv Vis sammendrag

  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. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Dowlatshah, Samira; Hansen, Frederik Andre; Zhou, Chen; Ramos-Payán, María; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2023). Electromembrane extraction of peptides based on hydrogen bond interactions. Analytica Chimica Acta. ISSN 0003-2670. 1275. doi: 10.1016/j.aca.2023.341610. Fulltekst i vitenarkiv Vis sammendrag

  Background Electromembrane extraction (EME) of peptides reported in the scientific literature involve transfer of net positively charged peptides from an aqueous sample, through a liquid membrane, and into an aqueous acceptor solution, under the influence of an electrical field. The liquid membrane comprises an organic solvent, containing an ionic carrier. The purpose of the ionic carrier is to facilitate peptide solvation in the organic solvent based on ionic interactions. Unfortunately, ionic carriers increase the conductivity of the liquid membrane; the current in the system increases, the electrolysis in sample and acceptor is accelerated, and the extraction system tend to be unstable and suffers from drifting pH. Results In the present work, a broad selection of organic solvents were tested as pure liquid membrane for EME of peptides, without ionic carrier. Several phosphates provided high mass transfer, and tri(pentyl) phosphate was selected since this solvent also provided high operational stability. Among 16 different peptides used as model analytes, tri(pentyl) phosphate extracted those with net charge +1 and with no more than two polar side chains. Tri(pentyl) phosphate served as a very strong hydrogen bond acceptor, while the protonated peptides were hydrogen bond donors. By such, hydrogen bonding served as the primary interactions responsible for mass transfer. Tri(pentyl) phosphate as liquid membrane, could exhaustively extract leu-enkephalin, met-enkephalin, and endomorphin from human blood plasma and detected by LC-MS/MS. Calibration curves were linear (r2 > 0.99) within a concentration range from 1 to 500 ng/mL, and a relative standard deviation within 12% was observed for precision studies. Significance The current experiments are important because they indicate that small peptides of low polarity may be extracted selectively in EME based on hydrogen bond interactions, in systems not suffering from electrolysis.

• 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. Fulltekst i vitenarkiv
• 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.

• 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. Fulltekst i vitenarkiv Vis sammendrag

  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), 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.

• 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), s. 5979–5989. doi: 10.1007/s00216-022-04161-w. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Eie, Linda Vårdal; Rye, Torstein Kige; Hansen, Frederik; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2021). Electromembrane extraction of peptides and amino acids – status and perspectives. Bioanalysis. ISSN 1757-6180. 13(4), s. 277–289. doi: 10.4155/bio-2020-0285. Fulltekst i vitenarkiv
• Rye, Torstein Kige; Martinovic, Gordana; Eie, Linda Vårdal; Hansen, Frederik Andre; Halvorsen, Trine Grønhaug & Pedersen-Bjergaard, Stig (2021). Electromembrane extraction of peptides using deep eutectic solvents as liquid membrane. Analytica Chimica Acta. ISSN 0003-2670. 1175. doi: 10.1016/j.aca.2021.338717. Fulltekst i vitenarkiv Vis sammendrag

  For the first time, we report electromembrane extraction (EME) of peptides using deep eutectic solvent (DES) as supported liquid membrane (SLM). DES were mixtures of coumarin, camphor, DL-menthol and thymol. Sixteen model peptides were extracted from 100 μL 50 mM phosphate buffer solution (pH 3.0), through the SLM, and into 100 μL acceptor solution consisting of 50 mM phosphoric acid (pH 1.8). EME was performed in 96-well format with 30 V to facilitate extraction of positively charged peptides. The model peptides comprised three to 13 amino acids, and differed significantly in terms of acid/base functionalities and polarity. We found pure DES to be inefficient for EME of peptides. However, with addition of a small amount of the ionic carrier di(2-ethylhexyl) phosphate (DEHP) to the DES, the extraction efficiency increased due to ionic interactions. With the most efficient SLM; coumarin and thymol mixed in molar ratio (1:2) with 2.0% (v/v) DEHP, average recovery after 15 min was 55%; five peptides were extracted with recovery > 80%, nine peptides with recoveries in the range 40–80%, and two peptides were not extracted (recovery < 5%). When extraction time was extended to 45 min, average extraction recovery increased to 83%. Extraction recoveries with DES were higher than previously reported in the literature for the same model peptides.

• 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. Fulltekst i vitenarkiv Vis sammendrag

  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. Fulltekst i vitenarkiv Vis sammendrag

  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. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Halvorsen, Trine Grønhaug; Levernæs, Maren Christin Stillesby & Rosting, Cecilie (2021). Matrix-Assisted Ionization and Tandem Mass Spectrometry Capabilities in Protein Biomarker Characterization—An Initial Study Using the Small Cell Lung Cancer Biomarker Progastrin Releasing Peptide as a Model Compound. Journal of the American Society for Mass Spectrometry. ISSN 1044-0305. 32(2), s. 611–614. doi: 10.1021/jasms.0c00336. Fulltekst i vitenarkiv Vis sammendrag

  This initial study evaluates vacuum matrix-assisted ionization (vMAI) mass spectrometry (MS) for identification and determination of tryptic peptides from the biomarker protein progastrin releasing peptide (ProGRP). Similar peptides and charge states were observed as in liquid chromatography (LC) electrospray ionization (ESI) MS. The prolonged ion duration in vMAI with similar charge states as in ESI was advantageous for determining the MS/MS fragmentation conditions compared to MAI. It is assumed that the vacuum ionization conditions lower the detection limits of the experiment. This may be the reason vMAI combined with high resolution MS enabled detection of tryptic peptides from more digested proteins than MAI selected reaction monitoring MS. Additionally, MAI ion mobility spectrometry MS (MAI-IMS–MS) was evaluated for differentiation of intact protein isoforms, successfully enabling differentiation of the isoforms by drift time selection. Examples are both shown for model proteins bovine serum albumin, cytochrome C, and lysozyme and the clinically relevant small cell lung cancer protein biomarker ProGRP, which exists in three isoforms. Coupling with the vacuum ionization conditions using a dedicated vacuum-probe source MAI enables information to be extracted readily as with conventional approaches, just faster.

• 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, s. 210–218. doi: 10.1016/j.aca.2020.10.022. Fulltekst i vitenarkiv
• 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), s. 3573–3582. doi: 10.1021/acs.jproteome.0c00258. Fulltekst i vitenarkiv
• 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), s. 222–235. Fulltekst i vitenarkiv Vis sammendrag

  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), s. 5476–5484. doi: 10.1039/d0ay01283a. Fulltekst i vitenarkiv
• Levernæs, Maren Christin Stillesby; Farhat, Bassem; Oulie, Inger; Abdullah, Sazan Salih; Paus, Elisabeth & Reubsaet, Leon [Vis alle 7 forfattere av denne artikkelen] (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), s. 34902–34911. doi: 10.1039/c9ra05071j. Fulltekst i vitenarkiv Vis sammendrag

  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, s. 764–770. doi: 10.1016/j.talanta.2018.12.013. Fulltekst i vitenarkiv
• 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, s. 56–65. doi: 10.1016/j.aca.2019.08.043. Fulltekst i vitenarkiv
• 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), s. 97–103. doi: 10.1039/c9ay01976f. Fulltekst i vitenarkiv
• Skjærvø, Øystein; Trimpin, Sarah & Halvorsen, Trine Grønhaug (2019). Matrix‐assisted ionization mass spectrometry in targeted protein analysis – An initial evaluation. Rapid Communications in Mass Spectrometry. ISSN 0951-4198. 32. doi: 10.1002/rcm.8437. Fulltekst i vitenarkiv Vis sammendrag

  Rationale Matrix‐assisted ionization (MAI) is a relatively new ionization technique for analysis by mass spectrometry (MS). The technique is simple and has been shown to be less influenced by matrix effects than e.g. electrospray ionization (ESI). These features are of interest in the targeted analysis of proteins from biological samples. Methods Targeted protein determination by MAI‐MS was evaluated using a triple quadrupole mass analyzer equipped with a stripped nanoESI source in selected reaction monitoring (SRM) mode. The proteins were analyzed using the bottom‐up approach with stable isotopic labeled peptides as internal standards (IS). The MAI matrix was 3‐nitrobenzonitrile dissolved in acetonitrile. Aqueous sample and matrix solution were mixed in a 1:3 volume ratio. One microlitre of the dried matrix/analyte sample was introduced into the inlet of the mass spectrometer where ionization commences. Results SRM settings established for ESI‐SRM‐MS of the peptides here investigated were applicable in MAI‐SRM‐MS for all evaluated peptides except one that is poorly soluble in water. Addition of IS provided efficient correction at most levels (relative standard deviation (RSD) ≤28% (except lowest digest level), r2 ≥ 0.995). This was also true for the more complex biological matrices, diluted urine (1:1; RSD = 20% a synthetic peptide, NLLGLIEAK) and diluted digested serum (1:100; RSD = 7% digested cytochrome C). Biological matrix influenced the signal intensity unless sufficiently diluted. Conclusions The results demonstrate that MAI‐SRM‐MS has promising potential in targeted protein determination by the bottom‐up approach because of its simplicity, ease of use, and speed. However, more data is needed to confirm the results prior to application in a clinical setting.

• Skjærvø, Øystein; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2018). Smart blood spots for whole blood protein analysis. The Analyst. ISSN 0003-2654. 143(13), s. 3184–3190. doi: 10.1039/c8an00317c. Fulltekst i vitenarkiv
• Rosting, Cecilie; Tran, Elin Vyvy; Gjelstad, Astrid & Halvorsen, Trine Grønhaug (2018). Determination of the Low-Abundant Protein Biomarker hCG from Dried Matrix Spots using Immunocapture and Nano Liquid Chromatography Mass Spectrometry. Journal of Chromatography B: Biomedical Sciences and Applications. ISSN 1387-2273. 1077-1078, s. 44–51. doi: 10.1016/j.jchromb.2018.01.016. Fulltekst i vitenarkiv Vis sammendrag

  Using LC-MS/MS for determination of low-abundance protein biomarkers from dried blood spots is challenging due to the combination of low biomarker levels (low pM-level) and small sample volumes (typically <50 μL). In the present paper it is demonstrated that use of state-of-the-art nano liquid chromatography triple quadrupole mass spectrometry in combination with immunoaffinity sample clean-up enable determination of the low abundance biomarker human chorionic gonadotropin (hCG) from four different biological matrices (whole blood, serum, plasma and urine) at its upper reference level (low pM). Detection limits for hCG was determined for all matrices from both commercially available non-soluble DBS sampling material (DMPK-C) and the water-soluble material carboxymethyl cellulose (CMC). The detection limits (S/N = 3) were ranging from 5.0 IU/L (14.5 pM; whole blood) to 10.5 IU/L (30.5 pM; urine) for DMPK-C and from 2.1 IU/L (6.1 pM; urine) to 6.4 IU/L (18.6 pM; plasma) for CMC. A brief evaluation was performed for both sampling materials using serum as matrix resulting in sufficient linearity (r2 ≥ 0.93, range 20–1000 IU/mL (58–2900 pM) for DMPK-C and 10–1000 IU/mL (29–2900 pM) for CMC), repeatability (RSD% = 13–31%) and accuracy (95–106%). To demonstrate the applicability of the method to real samples, a serum sample from a patient previously diagnosed with cancer was also analyzed using both sampling materials. The concentration levels found using the two materials were similar (5280± 595 IU/L (15,312 ± 1726 pM, n = 3) in the DMPK-C spot and 5060 ± 430 IU/L (14,674 ± 1247 pM, n = 3) in the CMC spot). All in all this demonstrated that the tools for determination of low abundance biomarkers at upper reference level from dried matrix spots now is available through a combination of immunoaffinity enrichment and state-of-the-art LC-MS/MS.

• Levernæs, Maren Christin Stillesby; Brandtzaeg, Ole Kristian; Amundsen, Sunniva Furre; Reubsaet, Leon; Lundanes, Elsa & Halvorsen, Trine Grønhaug [Vis alle 7 forfattere av denne artikkelen] (2018). Selective Fishing for Peptides with Antibody Immobilized Acrylate Monoliths, Coupled Online with NanoLC-MS. Analytical Chemistry. ISSN 0003-2700. 90, s. 13860–13860. doi: 10.1021/acs.analchem.8b00935. Fulltekst i vitenarkiv
• Andersen, Ida Kristine Lysgaard; Rosting, Cecilie; Gjelstad, Astrid & Halvorsen, Trine Grønhaug (2018). Volumetric absorptive MicroSampling vs. other blood sampling materials in LC-MS-based protein analysis - preliminary investigations. Journal of Pharmaceutical and Biomedical Analysis. ISSN 0731-7085. 156, s. 239–246. doi: 10.1016/j.jpba.2018.04.036. Fulltekst i vitenarkiv Vis sammendrag

  The aim was to evaluate the performance of different microsampling materials in LC–MS-based protein analysis. The evaluated materials were the Volumetric Absorptive MicroSampling (VAMS) device, the pure cellulose sampling material (DMPK-C) and the water-soluble material (carboxymethyl cellulose, CMC), with the main emphasis on VAMS. Six proteins with different physicochemical properties were used as model proteins. A quick and generic sample preparation consisting of extraction/dissolution of the blood spot, tryptic digestion and subsequent matrix precipitation was applied prior to analysis. The recovery from VAMS, compared to DMPK-C, was dependent on the protein analyte: Lower recovery compared to DMPK-C was seen for β-lactoglobulin and myoglobin (74% and 80%, respectively) while higher recovery compared to DMPK-C was seen for cytochrome c and albumin (149% and 197%, respectively). The recovery from CMC was comparable to the recovery from DMPK-C for all proteins except for cytochrome c (76%). Hematocrit bias was evaluated for DMPK-C and VAMS, and the blood hematocrit influenced the protein analysis from both the materials. A preliminary evaluation was performed for VAMS: The correlation coefficient (R2 ≥ 0.983), the accuracy (71–101%) and the precision (RSD ≤ 20%) were determined for blood spiked with the six model proteins.

• 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, s. 132–139. doi: 10.1016/j.trac.2017.08.009. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Rosting, Cecilie; Gjelstad, Astrid & Halvorsen, Trine Grønhaug (2017). Expanding the knowledge on dried blood spots and LC-MS-based protein analysis: two different sampling materials and six protein targets. Analytical and Bioanalytical Chemistry. ISSN 1618-2642. 409(13), s. 3383–3392. doi: 10.1007/s00216-017-0280-3. Fulltekst i vitenarkiv
• 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), s. 5631–5643. doi: 10.1007/s00216-017-0520-6. Fulltekst i vitenarkiv Vis sammendrag

  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.

• 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. Fulltekst i vitenarkiv Vis sammendrag

  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, s. 51–60. doi: 10.1016/j.jchromb.2017.03.044. Fulltekst i vitenarkiv Vis sammendrag

  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), s. 3837–3847. doi: 10.1039/c7an01075c. Fulltekst i vitenarkiv Vis sammendrag

  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, s. 19–26. doi: 10.1016/j.chroma.2016.09.069. Fulltekst i vitenarkiv
• 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). Fulltekst i vitenarkiv Vis sammendrag

  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.

• Rosting, Cecilie; Sæ, Christine Østvik; Gjelstad, Astrid & Halvorsen, Trine Grønhaug (2016). Evaluation of water-soluble DBS for small proteins: A conceptual study using insulin as a model analyte. Bioanalysis. ISSN 1757-6180. 8(10), s. 1051–1065. doi: 10.4155/bio-2016-0002. Fulltekst i vitenarkiv
• 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, s. 155–161. doi: 10.1016/j.jpba.2016.02.011. Fulltekst i vitenarkiv
• 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), s. 7378–7391. doi: 10.1007/s00216-016-9818-z. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Ask, Kristine Skoglund; Turgay, Bardakci; Parmer, Marthe Petrine; Halvorsen, Trine Grønhaug; Øiestad, Elisabeth Leere & Pedersen-Bjergaard, Stig [Vis alle 7 forfattere av denne artikkelen] (2016). Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma. Journal of Pharmaceutical and Biomedical Analysis. ISSN 0731-7085. 129, s. 229–236. doi: 10.1016/j.jpba.2016.07.011. Fulltekst i vitenarkiv
• Pedersen-Bjergaard, Stig; Gjelstad, Astrid & Halvorsen, Trine Grønhaug (2015). Sample preparation. I Hansen, Steen Honoré & Pedersen-Bjergaard, Stig (Red.), Bioanalysis of pharmaceuticals : sample preparation, separation techniques and mass spectrometry. Wiley-Blackwell. ISSN 9781118716823. s. 73–121. Fulltekst i vitenarkiv
• Kaupang, Åsmund; Hildonen, Siri; Halvorsen, Trine Grønhaug; Mortén, Magnus; Vik, Anders & Hansen, Trond Vidar (2015). Involvement of covalent interactions in the mode of action of PPARβ/δ antagonists. RSC Advances. 5(93), s. 76483–76490. doi: 10.1039/c5ra15707b. Fulltekst i vitenarkiv
• Kaupang, Åsmund; Paulsen, Steinar Martin; Steindal, Calin Constantin; Ravna, Aina Westrheim; Sylte, Ingebrigt & Halvorsen, Trine Grønhaug [Vis alle 8 forfattere av denne artikkelen] (2015). Synthesis, biological evaluation and molecular modeling studies of the PPARβ/δ antagonist CC618. European Journal of Medicinal Chemistry. ISSN 0223-5234. 94, s. 229–236. doi: 10.1016/j.ejmech.2015.03.006. Fulltekst i vitenarkiv Vis sammendrag

  Abstract: Herein, we describe the synthesis, biological evaluation and molecular docking of the selective PPARb/ d antagonist (4-methyl-2-(4-(trifluoromethyl)phenyl)-N-(2-(5-(trifluoromethyl)-pyridin-2-ylsulfonyl) ethyl)thiazole-5-carboxamide)), CC618. Results from in vitro luciferase reporter gene assays against the three known human PPAR subtypes revealed that CC618 selectively antagonizes agonist-induced PPARb/ d activity with an IC50 ¼ 10.0 mM. As observed by LC-MS/MS analysis of tryptic digests, the treatment of PPARb/d with CC618 leads to a covalent modification of Cys249, located centrally in the PPARb/d ligand binding pocket, corresponding to the conversion of its thiol moiety to a 5-trifluoromethyl-2- pyridylthioether. Finally, molecular docking is employed to shed light on the mode of action of the antagonist and its structural consequences for the PPARb/d ligand binding pocket.

• Rosting, Cecilie; Gjelstad, Astrid & Halvorsen, Trine Grønhaug (2015). Water-Soluble Dried Blood Spot in Protein Analysis: A Proof-of-Concept Study. Analytical Chemistry. ISSN 0003-2700. 87(15), s. 7918–7924. doi: 10.1021/acs.analchem.5b01735. Fulltekst i vitenarkiv
• 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), s. 12291–12298. doi: 10.1021/ac503559c. Fulltekst i vitenarkiv
• 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), s. 6983–6992. doi: 10.1021/ac500986t. Fulltekst i vitenarkiv
• 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), s. 2733–2738. doi: 10.1007/s00216-014-7634-x. Fulltekst i vitenarkiv
• Lund, Hanne; Paus, Elisabeth; Berger, Peter; Stenman, Ulf-Håkan; Torcellini, Tamara & Halvorsen, Trine Grønhaug [Vis alle 7 forfattere av denne artikkelen] (2014). Epitope analysis and detection of human chorionic gonadotropin (hCG) variants by monoclonal antibodies and mass spectrometry. Tumour Biology. ISSN 1010-4283. 35(2), s. 1013–1022. doi: 10.1007/s13277-013-1135-y. Fulltekst i vitenarkiv
• 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), s. 2031–2041. doi: 10.1002/pmic.201300479. Fulltekst i vitenarkiv
• Vestrheim, Anne Cathrine; Moen, Anders; Egge-Jacobsen, Wolfgang; Reubsaet, Leon; Halvorsen, Trine Grønhaug & Bratlie, Diane Bryant [Vis alle 8 forfattere av denne artikkelen] (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), s. 76–91. doi: 10.1002/iid3.22. Fulltekst i vitenarkiv

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• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2020). 100 years of hCG. Elsevier. ISBN 9780128200506. 331 s. Fulltekst i vitenarkiv
• Pedersen-Bjergaard, Stig; Gammelgaard, Bente & Halvorsen, Trine Grønhaug (2019). Introduction to Pharmaceutical Analytical Chemistry, 2nd Edition. Wiley-Blackwell. ISBN 9781119362722. Fulltekst i vitenarkiv

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• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2023). Smart sampling: next-generation DBS samplers for LC-MS based protein analysis. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2023). Next generation dried blood spot samplers for simplified LC-MS/MS analysis of proteins. Fulltekst i vitenarkiv
• Malerød-Fjeld, Helle & Halvorsen, Trine Grønhaug (2023). The 25th Norwegian symposium on chromatography. Analytical Science Advances (ASA). 4(7-8), s. 233–235. doi: 10.1002/ansa.202300035. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2023). Smart samplers for simplified LC-MS/MS analysis of proteins. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2023). Smart samplers for mass spectrometric bioanalysis of proteins. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2022). Syntetisk antistoff forenklar analyse av blodprøve. [Journal]. Forskning.no. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2022). Smart samplers in mass spectrometric bioanalysis of proteins. Fulltekst i vitenarkiv
• Vik, Anders; Halvorsen, Trine Grønhaug; Primdahl, Karoline Gangestad; Hansen, Trond Vidar; Wangensteen, Helle & Reubsaet, Leon [Vis alle 7 forfattere av denne artikkelen] (2022). Grunnkurs i farmasøytisk kjemi på UiO moderniseres kontinuerlig. Norsk Farmaceutisk Tidsskrift (NFT). ISSN 0029-1935. s. 33–34. Fulltekst i vitenarkiv
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). Smart Samplers for LC-MS based protein analysis. Fulltekst i vitenarkiv
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). Smart Samplers for LC-MS based protein analysis. Fulltekst i vitenarkiv
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). Smart samplers for LC-MS based protein analysis. Fulltekst i vitenarkiv
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). Smart samplers for LC-MS based protein analysis. Fulltekst i vitenarkiv
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2022). Smart Sampler for LC-MS based protein analysis. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug & Reubsaet, Leon (2021). Advances in sampling and sample preparation for mass spectrometric determination of protein biomarkers. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug (2020). Mass spectrometric determination of protein biomarkers from biological matrices – Advances in sampling and sample preparation. Fulltekst i vitenarkiv
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2019). Affinity based sample clean-up prior to LC-MS/MS for determination of low abundance proteins. Fulltekst i vitenarkiv
• Skjærvø, Øystein; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2019). Smart blood-spot sampling for MS based bottom-up protein analysis. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug (2019). Dried Blood Spots and Mass Spectrometry in Analysis of Protein Biomarkers and Biopharmaceuticals. Fulltekst i vitenarkiv
• Skjærvø, Øystein; Halvorsen, Trine Grønhaug & Reubsaet, Leon (2019). Smart blood spot sampling for MS tased bottom-up protein analysis. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug (2018). Analysis of proteins from biological matrices using affinity based sample clean-up and LC-MS/MS. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug (2018). Application of novel ionization techniques in determination of clinically relevant proteins. Fulltekst i vitenarkiv
• Brandtzaeg, Ole Kristian; Levernæs, Maren Christin Stillesby; Halvorsen, Trine Grønhaug; Reubsaet, Leon; Røen, Bent Tore & Vehus, Tore [Vis alle 10 forfattere av denne artikkelen] (2018). Online sample processing systems for targeted nano liquid chromatography mass spectrometry bioanalysis. Fulltekst i vitenarkiv
• Skjærvø, Øystein; Pilarova, Veronika; Khalikova, Maria; Reubsaet, Leon; Halvorsen, Trine Grønhaug & Gjelstad, Astrid [Vis alle 10 forfattere av denne artikkelen] (2018). Those Who Can, Teach: Pharma Stars. The Analytical Scientist. ISSN 2051-4077. 1118, s. 40–45. Fulltekst i vitenarkiv
• Skjærvø, Øystein; Pilarova, Veronika; Khalikova, Maria; Reubsaet, Leon; Halvorsen, Trine Grønhaug & Gjelstad, Astrid [Vis alle 10 forfattere av denne artikkelen] (2018). Those Who Can, Teach: Pharma Stars. The Analytical Scientist. ISSN 2051-4077. 1118, s. 40–45. Fulltekst i vitenarkiv
• 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), s. 1607–1608. doi: 10.1007/s00216-017-0797-5. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug (2016). Hva er viktig når jeg lager mobilfase til LC-MS? Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Reubsaet, Leon & Halvorsen, Trine Grønhaug (2015). Analysis of Peptide and Protein Drugs in Biological Fluids. I Hansen, Steen Honoré & Pedersen-Bjergaard, Stig (Red.), Bioanalysis of pharmaceuticals : sample preparation, separation techniques and mass spectrometry. Wiley-Blackwell. ISSN 9781118716823. s. 261–282. Fulltekst i vitenarkiv
• Ask, Kristine Skoglund; Halvorsen, Trine Grønhaug; Pedersen-Bjergaard, Stig & Gjelstad, Astrid (2015). Phospholipid removal from human plasma samples by Parallel Artificial Liquid Membrane Extraction. Fulltekst i vitenarkiv
• 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. Fulltekst i vitenarkiv
• Kaupang, Åsmund; Hildonen, Siri; Halvorsen, Trine Grønhaug; Mortén, Magnus; Vik, Anders & Hansen, Trond Vidar (2015). Investigations on the covalent antagonistic modulation of PPARβ/δ. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug (2014). Mobilfaser i HPLC - Ti uvaner du bør unngå. Fulltekst i vitenarkiv
• Halvorsen, Trine Grønhaug; Lund, Hanne & Reubsaet, Leon (2014). Immunoaffinity + LC-MS/MS: An alternative approach for antibody selectivity evaluation? Fulltekst i vitenarkiv
• Vehus, Tore; Wilson, Steven Ray Haakon; Lundanes, Elsa; Krauss, Stefan; Halvorsen, Trine Grønhaug & Morth, Jens Preben (2018). Nano Liquid Chromatography-Tandem Mass Spectrometry Platforms for Determination of Low Abundant Wnt/Beta-Catenin Proteins in Cancer Research. Universitetet i Oslo. Fulltekst i vitenarkiv

Se alle arbeider i NVA