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Publications (10 of 21) Show all publications
Hober, A., Edfors, F., Ryaboshapkina, M., Malmqvist, J., Rosengren, L., Percy, A. J., . . . Miliotis, T. (2019). Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay. Molecular & Cellular Proteomics, 18(12), 2433-2446
Open this publication in new window or tab >>Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay
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2019 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, no 12, p. 2433-2446Article in journal (Refereed) Published
Abstract [en]

Stable isotope-labeled standard (SIS) peptides are used as internal standards in targeted proteomics to provide robust protein quantification, which is required in clinical settings. However, SIS peptides are typically added post trypsin digestion and, as the digestion efficiency can vary significantly between peptides within a protein, the accuracy and precision of the assay may be compromised. These drawbacks can be remedied by a new class of internal standards introduced by the Human Protein Atlas project, which are based on SIS recombinant protein fragments called SIS PrESTs. SIS PrESTs are added initially to the sample and SIS peptides are released on trypsin digestion. The SIS PrEST technology is promising for absolute quantification of protein biomarkers but has not previously been evaluated in a clinical setting. An automated and scalable solid phase extraction workflow for desalting and enrichment of plasma digests was established enabling simultaneous preparation of up to 96 samples. Robust high-precision quantification of 13 apolipoproteins was achieved using a novel multiplex SIS PrEST-based LC-SRM/MS Tier 2 assay in non-depleted human plasma. The assay exhibited inter-day coefficients of variation between 1.5% and 14.5% (median = 3.5%) and was subsequently used to investigate the effects of omega-3 carboxylic acids (OM3-CA) and fenofibrate on these 13 apolipoproteins in human plasma samples from a randomized placebo-controlled trial, EFFECT I (NCT02354976). No significant changes were observed in the OM3-CA arm, whereas treatment with fenofibrate significantly increased apoAII and reduced apoB, apoCI, apoE and apoCIV levels. The reduction in apoCIV following fenofibrate treatment is a novel finding. The study demonstrates that SIS PrESTs can facilitate the generation of robust multiplexed biomarker Tier 2 assays for absolute quantification of proteins in clinical studies. Applications of LC-SRM in clinical research are still limited. SIS PrEST are a novel class of standards added prior to trypsinization. We have developed a semi-automated sample preparation workflow and a SIS PrEST LC-SRM/MS Tier 2 assay for absolute quantification of 13 apolipoproteins in human plasma and applied it on clinical samples from the EFFECT I study. We demonstrate, for the first time, that SIS PrEST can be applied for exploratory biomarker research in clinical settings and capture drug effects.

Place, publisher, year, edition, pages
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2019
Keywords
Clinical trials, assay development, targeted mass spectrometry, selected reaction monitoring, absolute quantification, apolipoproteins, fenofibrate and omega-3 carboxylic acids, NAFLD, SIS PrEST
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-266286 (URN)10.1074/mcp.RA119.001765 (DOI)000501288700007 ()31591263 (PubMedID)2-s2.0-85075958078 (Scopus ID)
Note

QC 20200108

Available from: 2020-01-08 Created: 2020-01-08 Last updated: 2020-01-10Bibliographically approved
Sahlström, P., Steen, J., Forsström, B., Titcombe, P., Stalesen, R., Nonhoff, U., . . . Grönwall, C. (2019). ANTI-MODIFIED PROTEIN AUTOANTIBODIES IN RA DISPLAY IMPORTANT PEPTIDE CROSS-REACTIVITY BUT YET PROTEIN RECOGNITION SELECTIVITY. Paper presented at Annual European Congress of Rheumatology (EULAR), JUN 12-15, 2019, Madrid, SPAIN. Annals of the Rheumatic Diseases, 78, 1439-1439
Open this publication in new window or tab >>ANTI-MODIFIED PROTEIN AUTOANTIBODIES IN RA DISPLAY IMPORTANT PEPTIDE CROSS-REACTIVITY BUT YET PROTEIN RECOGNITION SELECTIVITY
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2019 (English)In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 78, p. 1439-1439Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
BMJ PUBLISHING GROUP, 2019
National Category
Rheumatology and Autoimmunity
Identifiers
urn:nbn:se:kth:diva-255454 (URN)10.1136/annrheumdis-2019-eular.6901 (DOI)000472207104269 ()
Conference
Annual European Congress of Rheumatology (EULAR), JUN 12-15, 2019, Madrid, SPAIN
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20190822

Available from: 2019-08-22 Created: 2019-08-22 Last updated: 2020-01-14Bibliographically approved
Steen, J., Forsström, B., Sahlström, P., Odowd, V., Israelsson, L., Krishnamurthy, A., . . . Malmstrom, V. (2019). Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell-Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis. Arthritis & Rheumatology, 71(2), 196-209
Open this publication in new window or tab >>Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell-Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis
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2019 (English)In: Arthritis & Rheumatology, ISSN 2326-5191, E-ISSN 2326-5205, Vol. 71, no 2, p. 196-209Article in journal (Refereed) Published
Abstract [en]

Objective Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross-reactivity. Methods IgG-secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full-length mAb (n = 93) and also as germline-reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated. Results Four unrelated anti-citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell-derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay. Conclusion These findings suggest that the high level of cross-reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.

Place, publisher, year, edition, pages
WILEY, 2019
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-244539 (URN)10.1002/art.40699 (DOI)000457458100004 ()30152202 (PubMedID)2-s2.0-85060756734 (Scopus ID)
Note

QC 20190404

Available from: 2019-04-04 Created: 2019-04-04 Last updated: 2019-04-04Bibliographically approved
Edfors, F., Forsström, B., Vunk, H., Kotol, D., Fredolini, C., Maddalo, G., . . . Uhlén, M. (2019). Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics. Journal of Proteome Research, 18(7), 2706-2718
Open this publication in new window or tab >>Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics
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2019 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 7, p. 2706-2718Article in journal (Refereed) Published
Abstract [en]

The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts, and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labeled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labeled recombinant proteins before trypsin digestion enables short digestion protocols (<= 60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINAS, and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2019
Keywords
targeted proteomics, stable isotope standards, mass spectrometry, protein quantification, recombinant proteins, protein fragment, trypsin digestion, spectral library, assay generation, peptide formation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-255390 (URN)10.1021/acs.jproteome.8b00924 (DOI)000474795500003 ()31094526 (PubMedID)2-s2.0-85067403932 (Scopus ID)
Note

QC 20190730

Available from: 2019-07-30 Created: 2019-07-30 Last updated: 2020-01-10Bibliographically approved
Drobin, K., Assadi, G., Hong, M.-G., Anggraeni Andersson, M., Fredolini, C., Forsström, B., . . . Halfvarson, J. (2019). Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci. Inflammatory Bowel Diseases, 25(2), 306-316
Open this publication in new window or tab >>Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci
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2019 (English)In: Inflammatory Bowel Diseases, ISSN 1078-0998, E-ISSN 1536-4844, Vol. 25, no 2, p. 306-316Article in journal (Refereed) Published
Abstract [en]

Few studies have investigated the blood proteome of inflammatory bowel disease (IBD). We characterized the serum abundance of proteins encoded at 163 known IBD risk loci and tested these proteins for their biomarker discovery potential. Based on the Human Protein Atlas (HPA) antibody availability, 218 proteins from genes mapping at 163 IBD risk loci were selected. Targeted serum protein profiles from 49 Crohns disease (CD) patients, 51 ulcerative colitis (UC) patients, and 50 sex- and age-matched healthy individuals were obtained using multiplexed antibody suspension bead array assays. Differences in relative serum abundance levels between disease groups and controls were examined. Replication was attempted for CD-UC comparisons (including disease subtypes) by including 64 additional patients (33 CD and 31 UC). Antibodies targeting a potentially novel risk protein were validated by paired antibodies, Western blot, immuno-capture mass spectrometry, and epitope mapping. By univariate analysis, 13 proteins mostly related to neutrophil, T-cell, and B-cell activation and function were differentially expressed in IBD patients vs healthy controls, 3 in CD patients vs healthy controls and 2 in UC patients vs healthy controls (q < 0.01). Multivariate analyses further differentiated disease groups from healthy controls and CD subtypes from UC (P < 0.05). Extended characterization of an antibody targeting a novel, discriminative serum marker, the laccase (multicopper oxidoreductase) domain containing 1 (LACC1) protein, provided evidence for antibody on-target specificity. Using affinity proteomics, we identified a set of IBD-associated serum proteins encoded at IBD risk loci. These candidate proteins hold the potential to be exploited as diagnostic biomarkers of IBD.

Place, publisher, year, edition, pages
Oxford University Press, 2019
Keywords
inflammatory bowel disease, affinity proteomics, LACC1
National Category
Gastroenterology and Hepatology
Identifiers
urn:nbn:se:kth:diva-249898 (URN)10.1093/ibd/izy326 (DOI)000462580900020 ()30358838 (PubMedID)2-s2.0-85059799081 (Scopus ID)
Note

QC 20190423

Available from: 2019-04-23 Created: 2019-04-23 Last updated: 2019-04-23Bibliographically approved
Uhlén, M., Karlsson, M. J., Hober, A., Svensson, A.-S., Scheffel, J., Kotol, D., . . . Sivertsson, Å. (2019). The human secretome. Science Signaling, 12(609), Article ID eaaz0274.
Open this publication in new window or tab >>The human secretome
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2019 (English)In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 12, no 609, article id eaaz0274Article in journal (Refereed) Published
Abstract [en]

The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immuno-assays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.

Place, publisher, year, edition, pages
NLM (Medline), 2019
National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:kth:diva-265462 (URN)10.1126/scisignal.aaz0274 (DOI)000499099300003 ()31772123 (PubMedID)2-s2.0-85075677906 (Scopus ID)
Note

QC 20191218

Available from: 2019-12-18 Created: 2019-12-18 Last updated: 2020-01-10Bibliographically approved
The, M., Edfors, F., Perez-Riverol, Y., Payne, S. H., Hoopmann, M. R., Palmblad, M., . . . Käll, L. (2018). A Protein Standard That Emulates Homology for the Characterization of Protein Inference Algorithms. Journal of Proteome Research, 17(5), 1879-1886
Open this publication in new window or tab >>A Protein Standard That Emulates Homology for the Characterization of Protein Inference Algorithms
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2018 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 17, no 5, p. 1879-1886Article in journal (Refereed) Published
Abstract [en]

A natural way to benchmark the performance of an analytical experimental setup is to use samples of known measured analytes are peptides and not the actual proteins one of the inherent problems of interpreting data is that the composition and see to what degree one can correctly infer the content of such a sample from the data. For shotgun proteomics, themselves. As some proteins share proteolytic peptides, there might be more than one possible causative set of proteins resulting in a given set of peptides and there is a need for mechanisms that infer proteins from lists of detected peptides. A weakness of commercially available samples of known content is that they consist of proteins that are deliberately selected for producing tryptic peptides that are unique to a single protein. Unfortunately, such samples do not expose any complications in protein inference. Hence, for a realistic benchmark of protein inference procedures, there is a need for samples of known content where the present proteins share peptides with known absent proteins. Here, we present such a standard, that is based on E. coli expressed human protein fragments. To illustrate the application of this standard, we benchmark a set of different protein inference procedures on the data. We observe that inference procedures excluding shared peptides provide more accurate estimates of errors compared to methods that include information from shared peptides, while still giving a reasonable performance in terms of the number of identified proteins. We also demonstrate that using a sample of known protein content without proteins with shared tryptic peptides can give a false sense of accuracy for many protein inference methods.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
Keywords
mass spectrometry, proteomics, protein inference, sample of known content, protein standard, proteofom, peptide, homology, benchmark
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:kth:diva-228270 (URN)10.1021/acs.jproteome.7b00899 (DOI)000431726700013 ()29631402 (PubMedID)2-s2.0-85046675818 (Scopus ID)
Note

QC 20180522

Available from: 2018-05-22 Created: 2018-05-22 Last updated: 2018-12-05Bibliographically approved
Jahn, M., Vialas, V., Karlsen, J., Maddalo, G., Edfors, F., Forsström, B., . . . Hudson, E. P. (2018). Growth of Cyanobacteria Is Constrained by the Abundance of Light and Carbon Assimilation Proteins. Cell reports, 25(2), 478-+
Open this publication in new window or tab >>Growth of Cyanobacteria Is Constrained by the Abundance of Light and Carbon Assimilation Proteins
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2018 (English)In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 25, no 2, p. 478-+Article in journal (Refereed) Published
Abstract [en]

Cyanobacteria must balance separate demands for energy generation, carbon assimilation, and biomass synthesis. We used shotgun proteomics to investigate proteome allocation strategies in the model cyanobacterium Synechocystis sp. PCC 6803 as it adapted to light and inorganic carbon (C-i) limitation. When partitioning the proteome into seven functional sectors, we find that sector sizes change linearly with growth rate. The sector encompassing ribosomes is significantly smaller than in E. coli, which may explain the lower maximum growth rate in Synechocystis. Limitation of light dramatically affects multiple proteome sectors, whereas the effect of C-i limitation is weak. Carbon assimilation proteins respond more strongly to changes in light intensity than to C-i. A coarse-grained cell economy model generally explains proteome trends. However, deviations from model predictions suggest that the large proteome sectors for carbon and light assimilation are not optimally utilized under some growth conditions and may constrain the proteome space available to ribosomes.

Place, publisher, year, edition, pages
et al., 2018
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-237095 (URN)10.1016/j.celrep.2018.09.040 (DOI)000446691400020 ()30304686 (PubMedID)2-s2.0-85054193580 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research Council Formas, 2015-939Swedish Research CouncilSwedish Foundation for Strategic Research , RBP14-0013
Note

QC 20181029

Available from: 2018-10-29 Created: 2018-10-29 Last updated: 2020-01-10Bibliographically approved
Zandian, A., Forsström, B., Häggmark-Månberg, A., Schwenk, J. M., Uhlén, M., Nilsson, P. & Ayoglu, B. (2017). Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy. Journal of Proteome Research, 16(3), 1300-1314
Open this publication in new window or tab >>Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy
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2017 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 16, no 3, p. 1300-1314Article in journal (Refereed) Published
Abstract [en]

The underlying molecular mechanisms of autoimmune diseases are poorly understood. To unravel the autoimmune processes across diseases, comprehensive and unbiased analyses of proteins targets recognized by the adaptive immune system are needed. Here we present an approach starting from high-density peptide arrays to characterize autoantibody repertoires and to identify new autoantigens. A set of ten plasma and serum samples from subjects with multiple sclerosis, narcolepsy, and without any disease diagnosis were profiled on a peptide array representing the whole proteome, hosting 2.2 million 12-mer peptides with a six amino acid lateral shift. On the basis of the IgG reactivities found on these whole-proteome peptide micro arrays, a set of 23 samples was then studied on a targeted array with 174 000 12-mer peptides of single amino acid lateral shift. Finally, verification of IgG reactivities was conducted with a larger sample set (n = 448) using the bead-based peptide microarrays. The presented workflow employed three different peptide microarray formats to discover and resolve the epitopes of human autoantibodies and revealed two potentially new autoantigens: MAP3K7 in multiple sclerosis and NRXN1 in narcolepsy. The presented strategy provides insights into antibody repertoire reactivity at a peptide level and may accelerate the discovery and validation of autoantigens in human diseases.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
Keywords
peptide microarrays, autoantibody profiling, epitope mapping narcolepsy, multiple sclerosis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-205512 (URN)10.1021/acs.jproteome.6b00916 (DOI)000395726200017 ()28121444 (PubMedID)2-s2.0-85014691057 (Scopus ID)
Funder
VINNOVAKnut and Alice Wallenberg Foundation
Note

QC 20170524

Available from: 2017-05-24 Created: 2017-05-24 Last updated: 2020-01-10Bibliographically approved
Edfors, F., Danielsson, F., Hallström, B. M., Käll, L., Lundberg, E., Ponten, F., . . . Uhlén, M. (2016). Gene-specific correlation of RNA and protein levels in human cells and tissues. Molecular Systems Biology, 12(10), Article ID 883.
Open this publication in new window or tab >>Gene-specific correlation of RNA and protein levels in human cells and tissues
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2016 (English)In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 12, no 10, article id 883Article in journal (Refereed) Published
Abstract [en]

An important issue for molecular biology is to establish whether transcript levels of a given gene can be used as proxies for the corresponding protein levels. Here, we have developed a targeted proteomics approach for a set of human non-secreted proteins based on parallel reaction monitoring to measure, at steady-state conditions, absolute protein copy numbers across human tissues and cell lines and compared these levels with the corresponding mRNA levels using transcriptomics. The study shows that the transcript and protein levels do not correlate well unless a gene-specific RNA-to-protein (RTP) conversion factor independent of the tissue type is introduced, thus significantly enhancing the predictability of protein copy numbers from RNA levels. The results show that the RTP ratio varies significantly with a few hundred copies per mRNA molecule for some genes to several hundred thousands of protein copies per mRNA molecule for others. In conclusion, our data suggest that transcriptome analysis can be used as a tool to predict the protein copy numbers per cell, thus forming an attractive link between the field of genomics and proteomics.

Place, publisher, year, edition, pages
Blackwell Publishing, 2016
Keywords
gene expression, protein quantification, targeted proteomics, transcriptomics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-196993 (URN)10.15252/msb.20167144 (DOI)000386948100001 ()2-s2.0-84992562628 (Scopus ID)
Note

QC 20161213

Available from: 2016-12-13 Created: 2016-11-28 Last updated: 2020-01-10Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-5248-8568

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