Analysis of 1508 Plasma Samples by Capillary-Flow Data-Independent Acquisition Profiles Proteomics of Weight Loss and Maintenance

Abstract

Comprehensive, high throughput analysis of the plasma proteome has the potential to enable holistic analysis of the health state of an individual. Based on our own experience and the evaluation of recent large-scale plasma mass spectrometry (MS) based proteomic studies, we identified two outstanding challenges: slow and delicate nano-flow liquid chromatography (LC) and irreproducibility of identification of data-dependent acquisition (DDA). We determined an optimal solution reducing these limitations with robust capillary-flow data-independent acquisition (DIA) MS. This platform can measure 31 plasma proteomes per day. Using this setup, we acquired a large-scale plasma study of the diet, obesity and genes dietary (DiOGenes) comprising 1508 samples. Proving the robustness, the complete acquisition was achieved on a single analytical column. Totally, 565 proteins (459 identified with two or more peptide sequences) were profiled with 74% data set completeness. On average 408 proteins (5246 peptides) were identified per acquisition (319 proteins in 90% of all acquisitions). The workflow reproducibility was assessed using 34 quality control pools acquired at regular intervals, resulting in 92% data set completeness with CVs for protein measurements of 10.9%.The profiles of 20 apolipoproteins could be profiled revealing distinct changes. The weight loss and weight maintenance resulted in sustained effects on low-grade inflammation, as well as steroid hormone and lipid metabolism, indicating beneficial effects. Comparison to other large-scale plasma weight loss studies demonstrated high robustness and quality of biomarker candidates identified. Tracking of nonenzymatic glycation indicated a delayed, slight reduction of glycation in the weight maintenance phase. Using stable-isotope-references, we could directly and absolutely quantify 60 proteins in the DIA.In conclusion, we present herein the first large-scale plasma DIA study and one of the largest clinical research proteomic studies to date. Application of this fast and robust workflow has great potential to advance biomarker discovery in plasma. Comprehensive, high throughput analysis of the plasma proteome has the potential to enable holistic analysis of the health state of an individual. Based on our own experience and the evaluation of recent large-scale plasma mass spectrometry (MS) based proteomic studies, we identified two outstanding challenges: slow and delicate nano-flow liquid chromatography (LC) and irreproducibility of identification of data-dependent acquisition (DDA). We determined an optimal solution reducing these limitations with robust capillary-flow data-independent acquisition (DIA) MS. This platform can measure 31 plasma proteomes per day. Using this setup, we acquired a large-scale plasma study of the diet, obesity and genes dietary (DiOGenes) comprising 1508 samples. Proving the robustness, the complete acquisition was achieved on a single analytical column. Totally, 565 proteins (459 identified with two or more peptide sequences) were profiled with 74% data set completeness. On average 408 proteins (5246 peptides) were identified per acquisition (319 proteins in 90% of all acquisitions). The workflow reproducibility was assessed using 34 quality control pools acquired at regular intervals, resulting in 92% data set completeness with CVs for protein measurements of 10.9%. The profiles of 20 apolipoproteins could be profiled revealing distinct changes. The weight loss and weight maintenance resulted in sustained effects on low-grade inflammation, as well as steroid hormone and lipid metabolism, indicating beneficial effects. Comparison to other large-scale plasma weight loss studies demonstrated high robustness and quality of biomarker candidates identified. Tracking of nonenzymatic glycation indicated a delayed, slight reduction of glycation in the weight maintenance phase. Using stable-isotope-references, we could directly and absolutely quantify 60 proteins in the DIA. In conclusion, we present herein the first large-scale plasma DIA study and one of the largest clinical research proteomic studies to date. Application of this fast and robust workflow has great potential to advance biomarker discovery in plasma. The circulatory system is the primary link between all parts of the body in both humans and animals. As such, it harbors the potential to indicate the health status of any key organ. Blood delivers necessary substances such as nutrients and oxygen to cells and transports metabolic waste products away from those same cells. It is also vital to the immune system-based host defense mechanisms. Blood consists of cells suspended in a liquid called plasma (1Sylvia M.S. Michael W. Human Biology, 14 edition. McGraw-Hill Education, New York, NY2015Google Scholar). Blood is well suited for diagnoses based on biomarkers, because it is readily obtainable with minimally invasive and repeatable sampling and large biobanks exist for retrospective analyses. Up to now, 3500 to 5000 proteins have been measured in plasma or serum (2Schwenk J.M. Omenn G.S. Sun Z. Campbell D.S. Baker M.S. Overall C.M. Aebersold R. Moritz R.L. Deutsch E.W. The Human Plasma Proteome Draft of 2017: Building on the Human Plasma PeptideAtlas from Mass Spectrometry and Complementary Assays.J. Proteome Res. 2017; 16: 4299-4310Crossref PubMed Scopus (130) Google Scholar, 3Keshishian H. Burgess M.W. Specht H. Wallace L. Clauser K.R. Gillette M.A. Carr S.A. Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.Nat. Protoc. 2017; 12: 1683-1701Crossref PubMed Scopus (88) Google Scholar). In addition, there are good arguments that all human proteins might be present in plasma to a certain extent given that blood flows through almost all organs of our body. However, not many more than 100 protein biomarkers from plasma have been cleared/approved by the Food and Drug Administration (FDA) 1The abbreviations used are: FDAFood and Drug AdministrationCVcoefficient of variationDDAdata-dependent acquisitionDIAdata-independent acquisitionFDRfalse discovery rateMS1peptide precursor survey scanMS2fragment ion scanCIDclinical investigation dayLCDlow-calorie dietWMDweight maintenance dietSISstable isotope standardLCliquid chromatographyRFradio frequencyMSmass spectrometryPTMpost-translational Modification. 1The abbreviations used are: FDAFood and Drug AdministrationCVcoefficient of variationDDAdata-dependent acquisitionDIAdata-independent acquisitionFDRfalse discovery rateMS1peptide precursor survey scanMS2fragment ion scanCIDclinical investigation dayLCDlow-calorie dietWMDweight maintenance dietSISstable isotope standardLCliquid chromatographyRFradio frequencyMSmass spectrometryPTMpost-translational Modification. (4Anderson N.L. Ptolemy A.S. Rifai N. The riddle of protein diagnostics: future bleak or bright?.Clin. Chem. 2013; 59: 194-197Crossref PubMed Scopus (45) Google Scholar). This contrasts with the high number of protein biomarker studies performed (5Hernández B. Parnell have proteomic biomarkers and PubMed Scopus Google Scholar). This that is to and or health biomarkers in plasma. Food and Drug Administration of data-dependent acquisition data-independent acquisition discovery peptide precursor survey ion clinical investigation weight maintenance isotope liquid chromatography mass spectrometry Modification. Food and Drug Administration of data-dependent acquisition data-independent acquisition discovery peptide precursor survey ion clinical investigation weight maintenance isotope liquid chromatography mass spectrometry Modification. discovery has to be a in many biomarker candidates have been a could be and are used in the for this are study large were or to large and quality (5Hernández B. 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