ORCID Profile
0000-0003-1862-8528
Current Organisations
SDU
,
SDU - University of Southern Denmark
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Publisher: American Chemical Society (ACS)
Date: 28-02-2008
DOI: 10.1021/PR800115F
Abstract: Serum protein profiling by mass spectrometry has achieved attention as a promising technology in oncoproteomics. We performed a systematic review of published reports on protein profiling as a diagnostic tool for breast cancer. The MEDLINE, EMBASE, and COCHRANE databases were searched for original studies reporting discriminatory protein peaks for breast cancer as either protein identity or as m/ z values in the period from January 1995 to October 2006. To address the important aspect of reproducibility of mass spectrometry data across different clinical studies, we compared the published lists of potential discriminatory peaks with those peaks detected in an original MALDI MS protein profiling study performed by our own research group. A total of 20 protein eptide profiling studies were eligible for inclusion in the systematic review. Only 3 reports included information on protein identity. Although the studies revealed a considerable heterogeneity in relation to experimental design, biological variation, preanalytical conditions, methods of computational data analysis, and analytical reproducibility of profiles, we found that 45% of peaks previously reported to correlate with breast cancer were also detected in our experimental study. Furthermore, 25% of these redetected peaks also showed a significant difference between cases and controls in our study. Thus, despite known problems related to reproducibility, we were able to demonstrate overlap in peaks between clinical studies indicating some convergence toward a set of common discriminating, reproducible peaks for breast cancer. These peaks should be further characterized for identification of the protein identity and validated as biomarkers for breast cancer.
Publisher: Wiley
Date: 03-2009
Abstract: Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and rigorous s le collection and s le handling protocols. The sensitivity of the MS analysis relies on the quality of the s le consequently, the blood s le preparation step is crucial to obtain pure and concentrated s les and enrichment of the proteins and peptides of interest. This review focuses on the serum s le preparation step prior to protein profiling by MALDI MS analysis, with particular focus on various SPE methods. The application of SPE techniques with different chromatographic properties such as RP, ion exchange, or affinity binding to isolate specific subsets of molecules (subproteomes) is advantageous for increasing resolution and sensitivity in the subsequent MS analysis. In addition, several of the SPE s le preparation methods are simple and scalable and have proven easy to automate for higher reproducibility and throughput, which is important in a clinical proteomics setting.
Publisher: Springer Science and Business Media LLC
Date: 14-08-2011
DOI: 10.1038/NSMB.2099
Publisher: Elsevier BV
Date: 08-2006
Publisher: American Chemical Society (ACS)
Date: 03-06-2010
DOI: 10.1021/PR100075X
Abstract: Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)-nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile-water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)-NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard s le (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 microg of mouse s le or 50 microg of Drosophila melanogaster s le, more than 1000 phosphorylation sites were identified in each study using IMAC-IMAC and LC-MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC-IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects.
Publisher: Wiley
Date: 24-05-2003
DOI: 10.1002/RCM.1960
Abstract: Serum profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) holds promise as a clinical tool for early diagnosis of cancer and other human diseases. S le preparation is key to achieving reproducible and well-resolved signals in MALDI-MS a prerequisite for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI-MS. We developed a simple protocol for serum profiling that combines a matrix mixture of 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid with miniaturized SPE and MALDI-MS. Functionalized membrane discs with hydrophobic, ion-exchange or chelating properties allowed reproducible MALDI mass spectra (m/z 1000-12,000) to be obtained from serum. In a proof-of-principle application, SPE with chelating material and MALDI-MS identified protein peaks in serum that had been previously reported for distinguishing a person diagnosed with breast cancer from a control. These preliminary results indicate that this simple SPE/MALDI-MS method for serum profiling provides a versatile and scalable platform for clinical proteomics.
Publisher: Wiley
Date: 10-01-2008
DOI: 10.1002/RCM.3364
Abstract: Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. S le preparation is a key issue in MALDI MS and the analysis of complex s les such as serum requires optimized, reproducible methods for handling and deposition of protein s les. Data acquisition in MALDI MS is also a critical issue, since heterogeneity of s le deposits leads to attenuation of ion signals in MALDI MS. In order to improve the robustness and reproducibility of MALDI MS for serum protein profiling we investigated a range of s le preparation techniques and developed a statistical method based on repeated analyses for evaluation of protein-profiling performance of MALDI MS. Two different solid-phase extraction (SPE) methods were investigated, namely custom-made microcolumns and commercially available magnetic beads. Using these two methods, nineteen different s le preparation methods for serum profiling by MALDI MS were systematically tested with regard to matrix selection, stationary phase, selectivity, and reproducibility. Microcolumns were tested with regard to chromatographic properties reversed phase (C8, C18, SDB-XC), ion-exchange (anion, weak cation, mixed-phase (SDB-RPS)) and magnetic beads were tested with regard to chromatographic properties reversed phase (C8) or affinity chromatography (Cu-IMAC). The reproducibility of each s le preparation method was determined by enumeration and analysis of protein signals that were detected in at least six out of nine spectra obtained by three triplicate analyses of one serum s le.A candidate for best overall performance as evaluated by the number of peaks generated and the reproducibility of mass spectra was found among the tested methods. Up to 418 reproducible peaks were detected in one cancer serum s le. These protein peaks can be part of a possible diagnostic profile, suggesting that this s le preparation method and data acquisition approach is suitable for large-scale analysis of serum s les for protein profiling.
Publisher: American Chemical Society (ACS)
Date: 28-02-2008
DOI: 10.1021/PR7007576
Abstract: Serum protein profiling by mass spectrometry is a promising method for early detection of cancer. We have implemented a combined strategy based on matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and statistical data analysis for serum protein profiling and applied it in a well-described breast cancer case-control study. A rigorous s le collection protocol ensured high quality specimen and reduced bias from preanalytical factors. Preoperative serum s les obtained from 48 breast cancer patients and 28 controls were used to generate MALDI MS protein profiles. A total of nine mass spectrometric protein profiles were obtained for each serum s le. A total of 533 common peaks were defined and represented a 'reference protein profile'. Among these 533 common peaks, we identified 72 peaks exhibiting statistically significant intensity differences ( p < 0.01) between cases and controls. A diagnostic rule based on these 72 mass values was constructed and exhibited a cross-validated sensitivity and specificity of approximately 85% for the detection of breast cancer. With this method, it was possible to distinguish early stage cancers from controls without major loss of sensitivity and specificity. We conclude that optimized serum s le handling and mass spectrometry data acquisition strategies in combination with statistical analysis provide a viable platform for serum protein profiling in cancer diagnosis.
Publisher: Wiley
Date: 09-2007
Abstract: The early transition of knowledge from highly specialised and sophisticated proteomics research to a erse community in need of know-how is a challenge that requires backing from advanced research centres and groups, and a coordinating body for the dissemination of this knowledge. The European Proteomics Association (EuPA) Education Committee signified this as a priority area when the EuPA was formed, and began its program to coordinate proteomics training and knowledge dissemination in 2006. This report serves as an update of our past activities and an announcement of upcoming events. Over the last year the EuPA Education Committee has coordinated or supported different educational activities including basic and advanced courses, a summer school, workshops and tutorials. A new programme of basic courses dubbed "Teaching the Teachers" has been initiated. These courses reach a larger, Europe wide, audience in a short timeframe, thus improving the opportunities for trainees of elementary proteomics techniques. Another important event has been the merger of the EuPA and HUPO (Human Proteome Organisation) Education Committees into a single one in order to combine ideas and ef for ts that will favour global education in proteomics.
Publisher: Elsevier BV
Date: 08-2020
Publisher: American Chemical Society (ACS)
Date: 16-06-2009
DOI: 10.1021/AC9008447
Abstract: Because of unparalleled sensitivity and tolerance to protein size, mass spectrometry (MS) has become a popular method for measuring the solution hydrogen (1H/2H) exchange (HX) of biologically relevant protein states. While incorporated deuterium can be localized to different regions by pepsin proteolysis of the labeled protein, the assignment of deuteriums to in idual residues is typically not obtained, thereby limiting a detailed understanding of HX and the dynamics of protein structure. Here we use gas-phase fragmentation of peptic peptides by electron transfer dissociation (ETD) to measure the HX of in idual amide linkages in the amyloidogenic protein beta2-microglobulin. A comparison of the deuterium levels of 60 in idual backbone amides of beta2-microglobulin measured by HX-ETD-MS analysis to the corresponding values measured by NMR spectroscopy shows an excellent correlation. The deuterium labeling pattern of beta2-microglobulin is retained in the gaseous fragment ions by employing mild declustering conditions for electrospray ionization. A recently developed model peptide is used to arrive at such ion source declustering conditions that prevent the occurrence of intramolecular gas-phase hydrogen (1H/2H) migration (i.e., hydrogen scrambling). This article demonstrates that ETD can be implemented in a mass spectrometric method to monitor the conformational dynamics of proteins in solution at single-residue resolution.
Publisher: Public Library of Science (PLoS)
Date: 13-09-2013
Publisher: Elsevier BV
Date: 04-2008
DOI: 10.1016/J.JPROT.2008.03.004
Abstract: Plans for the European Proteomics Association (EuPA) were conceived and established during 2004 and 2005, and culminated in the formal inception of the organisation during the 4th HUPO World Congress held in Munich in 2005. The mission from the outset has been three-tiered and is to: i) strengthen the national Proteomics organizations in their efforts ii) to co-ordinate and provide educational programs, and iii) to advance the networking of scientists through meetings, workshops and student exchange. Linked to the mission were objectives to emphasise the benefits and contributions of Proteomics to biological and industrial researchers, the general public and science policy makers in Europe. In addition, the EuPA set out to promote scientific exchange for all applications and technology development related to Proteomics, and coordinate joint activities of national Proteomics societies at the European level. To achieve these tasks an organisational structure was conceived whereby four Activity Committees (Conferences/Communications, Education, EuPA-HUPO-Interactions and Funding) were implemented and a General Council consisting of all member countries. The remarkable rise and progress the EuPA has achieved in this small time frame is reported here.
Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1016/J.JPROT.2012.03.023
Abstract: Trans fatty acid intake has been correlated to an unfavorable plasma lipoprotein profile and an increased cardiovascular disease risk. The present study aimed to identify a plasma protein biomarker panel related to human intake of elaidic acid. The human liver cell line HepG2-SF was used as a model system, and the cells were maintained for seven days in serum-free medium containing 100 μM elaidic acid (trans∆9-C18:1), oleic acid (cis∆9-C18:1) or stearic acid (C18:0). The secretomes were analyzed by stable isotope labeling of amino acids in cell culture (SILAC), difference in gel electrophoresis (DIGE) and gene expression microarray analysis. Twelve proteins were found to be differentially regulated based on SILAC data (>1.3 fold change, P-value 1.3 fold change, P-value 1.3 fold change, P-value<0.01) following the addition of elaidic acid compared to oleic acid or stearic acid. The results revealed that 37 proteins were regulated specifically in response to elaidic acid exposure, and nine of these proteins were confirmed to be regulated in this manner by using selected reaction monitoring mass spectrometry.
Publisher: Springer Science and Business Media LLC
Date: 14-02-2018
Publisher: American Chemical Society (ACS)
Date: 29-10-2010
DOI: 10.1021/AC101889B
Abstract: The application of electron-transfer dissociation (ETD) to obtain single-residue resolution in hydrogen exchange-mass spectrometry (HX-MS) experiments has recently been demonstrated. For such measurements, it is critical to ensure that the level of gas-phase hydrogen scrambling is negligible. Here we utilize the abundant loss of ammonia upon ETD of peptide ions as a universal reporter of positional randomization of the exchangeable hydrogens (hydrogen scrambling) during HX-ETD experiments. We show that the loss of ammonia from peptide ions proceeds without depletion of deuterium when employing optimized mild electrospray ion source settings for the HX-ETD analysis of a selectively labeled model peptide and peptides derived from fully labeled β(2)-microglobulin. Hydrogen scrambling, as induced by excessive vibrational excitation of peptide ions during harsh declustering conditions, is easily detected by a depletion of deuterium when deuterated ammonia is lost from peptides during ETD. This straightforward method requires no modifications to the experimental workflow and has the great advantage that the occurrence of hydrogen scrambling can be directly detected in the actual peptides analyzed in the HX-ETD experiment.
Publisher: Public Library of Science (PLoS)
Date: 13-03-2015
Publisher: Elsevier BV
Date: 05-2008
Publisher: American Chemical Society (ACS)
Date: 27-04-2006
DOI: 10.1021/PR0504539
Abstract: Protein complexes are dynamic entities identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins associated with the translation initiation eIF4F complex that may affect complex formation were identified and quantitated under distinct growth conditions. Site-specific phosphorylation of eIF4E and eIF4G and elevated levels of eIF4G:eIF4E complexes in phorbol ester treated HEK293 cells, and in serum-starved tumorigenic human mesenchymal stromal cells, attested to their activated translational states. The WD-repeat, scaffolding-protein Gemin5 was identified as a novel eIF4E binding partner, which interacted directly with eIF4E through a motif (YXXXXLPhi) present in a number of eIF4E-interacting partners. Elevated levels of Gemin5:eIF4E complexes were found in phorbol ester treated HEK293 cells. Gemin5 and eIF4E co-localized to cytoplasmic P-bodies in human osteosarcoma U2OS cells. Interaction between eIF4E and Gemin5 and their co-localization to the P-bodies, may serve to recruit capped mRNAs to these RNP complexes, for functions related to RNP assembly, remodeling and/or transition from active translation to mRNA degradation. Our results demonstrate that our quantitative proteomic strategy can be applied to the identification and quantitation of protein complex components in human cells grown under different conditions.
Publisher: Elsevier BV
Date: 12-2007
DOI: 10.1016/J.ABB.2007.09.015
Abstract: Among isoflavonoid beta-glucosidases from Dalbergia species, that from Dalbergia nigrescens hydrolyzes isoflavonoid-7-O-beta-D-apiosyl-1,6-beta-D-glucosides more efficiently, while Dalbergia cochinchinensis beta-glucosidase (dalcochinase) hydrolyzes its rotenoid glycoside substrate, dalcochinin beta-d-glucoside (I), more efficiently. A cDNA encoding a glycosylated beta-glucosidase with 81% identity with dalcochinase was cloned from D. nigrescens seeds, and its protein (Dnbglu2) expressed in Pichia pastoris. Purified Dnbglu2 hydrolyzed the D. nigrescens natural substrates dalpatein 7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (II) and dalnigrein 7-O-beta-d-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (III) at 400- and 5000-fold higher catalytic efficiency (k(cat)/K(m)) than I. Dalcochinase was mutated at two amino acid residues, A454S and E455G, that are homologous to previously described substrate binding residues and differ from the corresponding residues in Dnbglu2. The double mutant showed 4- and 6.8-fold increases in relative activity toward II and III, respectively. However, this activity was only 3% that of Dnbglu2 beta-glucosidase, indicating other determinants are important for isoflavonoid diglycoside hydrolysis.
Publisher: Wiley
Date: 03-2010
Abstract: Characterization of low microgram levels of glycoprotein remains a challenge due to extensive heterogeneity of the conjugated N ‐glycans at each in idual glycosylation site. We present an optimized, sensitive workflow for glycopeptide isolation and characterization that exploits the complementary features of RP (Poros R2) and hydrophilic (zwitter‐ionic hydrophilic interaction chromatography) chromatographic resins. The glycopeptide analysis workflow was applied to human β2‐glycoprotein I (β2‐GPI, apolipoprotein H), which contains multiple N ‐glycosylation sites. Conditions for rapid proteolytic digestion of β2‐GPI using low‐specificity proteases were optimized to detect β2‐GPI glycopeptides by MS. We demonstrate the importance of ensuring sufficient column capacity of both hydrophobic and hydrophilic stationary phases for optimal glycoprofiling by MS. The enriched glycopeptides were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi‐ and tri‐antennary complex type glycans, were characterized at three N ‐glycosylation sites, namely Asn‐143, Asn‐174 and Asn‐234, of β2‐GPI. Further exploration of the complementary nature of RP and HILIC stationary phases for glycopeptide isolation prior to MS analysis may eventually enable systematic analysis of complex glycoprotein s les in functional proteomic research and advance our understanding of the biological role of protein glycosylation.
Publisher: Elsevier BV
Date: 06-2012
DOI: 10.1016/J.JPROT.2012.02.007
Abstract: The focus of this systematic review is to give an overview of the current status of clinical protein profiling studies using MALDI and SELDI MS platforms in the search for ovarian cancer biomarkers. A total of 34 profiling studies were qualified for inclusion in the review. Comparative analysis of published discriminatory peaks to peaks found in an original MALDI MS protein profiling study was made to address the key question of reproducibility across studies. An overlap was found despite substantial heterogeneity between studies relating to study design, biological material, pre-analytical treatment, and data analysis. About 47% of the peaks reported to be associated to ovarian cancer were also represented in our experimental study, and 34% of these redetected peaks also showed a significant difference between cases and controls in our study. Thus, despite known problems related to reproducibility an overlap in peaks between clinical studies was demonstrated, which indicate convergence toward a set of common discriminating, reproducible peaks for ovarian cancer. The potential of the discriminating protein peaks for clinical use as ovarian cancer biomarkers will be discussed and evaluated. This article is part of a Special Issue entitled: Proteomics: The clinical link.
Publisher: Springer Science and Business Media LLC
Date: 06-03-2014
DOI: 10.1038/NCOMMS4394
Publisher: Elsevier BV
Date: 04-2000
Publisher: American Chemical Society (ACS)
Date: 24-12-2008
DOI: 10.1021/JA805573H
Abstract: Mass spectrometry is routinely applied to measure the incorporation of deuterium into proteins and peptides. The exchange of labile, heteroatom-bound hydrogens is mainly used to probe the structural dynamics of proteins in solution, e.g., by hydrogen-exchange mass spectrometry, but also to study the gas-phase structure and fragmentation mechanisms of polypeptide ions. Despite considerable effort in recent years, there is no widely established mass spectrometric method to localize the incorporated deuterium to single amino acid residues, and typically, only the overall deuterium content of peptides or proteins is obtained. The main reason for this is that CID and related techniques induce intramolecular migration of hydrogens ("hydrogen scrambling") upon vibrational excitation of the even-electron precursor ion, thus randomizing the positional distribution of the incorporated deuterium atoms before fragmentation. In contrast, decomposition of radical gas-phase peptide cations upon electron capture dissociation was recently demonstrated to proceed with a very low level of amide hydrogen scrambling. Employing model peptides developed to enable sensitive detection of hydrogen scrambling, we show in the present study that electron transfer dissociation in a 3D-quadrupole ion trap retains the site-specific solution-phase deuterium incorporation pattern and allows for localization of incorporated deuterium with single residue resolution. Furthermore, we exploit this finding to monitor how collisional activation induces proton mobility in a gaseous peptide ion at various levels of vibrational excitation.
Publisher: Elsevier BV
Date: 02-2021
Publisher: Elsevier BV
Date: 03-2017
DOI: 10.1016/J.JPROT.2016.12.019
Abstract: Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95°C) and increased sensitivity achieved by reduction of analyte losses during s le preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein s les. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14Da), an oxidised form of methionine - aspartate semialdehyde (-32Da) - and decarboxylated glutamic acid and aspartic acid (-30Da). Proteomic tools provide a promising way to decode disease mechanisms at the protein level and help to understand how carbonylation affects protein structure and function. The challenge for future research is to identify the type and nature of oxidised residues to gain a deeper understanding of the mechanism(s) governing carbonylation in cells and organisms and assess their role in disease.
No related grants have been discovered for Ole Nørregaard Jensen.