Roland Bruderer

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics.We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics.Utilizing HRM, we profiled acetaminophen (APAP) 1The abbreviations used are:APAPacetaminophenATPadenosine triphosphateCVcoefficient of variationDIAdata-independent acquisitionDDAdata-dependent acquisitionFDRfalse discovery rateHRMhyper reaction monitoringiRTindexed retention timeNAPQIN-acetyl-p-benzoquinone imineSRMselected reaction monitoring; DIA with 32 sequential windows of 25 Dalton width. treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD).Our findings imply that DIA should be the preferred method for quantitative protein profiling. The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP) 1The abbreviations used are:APAPacetaminophenATPadenosine triphosphateCVcoefficient of variationDIAdata-independent acquisitionDDAdata-dependent acquisitionFDRfalse discovery rateHRMhyper reaction monitoringiRTindexed retention timeNAPQIN-acetyl-p-benzoquinone imineSRMselected reaction monitoring; DIA with 32 sequential windows of 25 Dalton width. treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). acetaminophen adenosine triphosphate coefficient of variation data-independent acquisition data-dependent acquisition false discovery rate hyper reaction monitoring indexed retention time N-acetyl-p-benzoquinone imine selected reaction monitoring; DIA with 32 sequential windows of 25 Dalton width. Our findings imply that DIA should be the preferred method for quantitative protein profiling. Quantitative mass spectrometry is a powerful and widely used approach to identify differentially abundant proteins, e.g. for proteome profiling and biomarker discovery (1Liu Y. Hittenhain R. Collins B. Aebersold R. Mass spectrometric protein maps for biomarker discovery and clinical research.Expert Rev. Mol. Diagn. 2013; 13: 811-825Crossref PubMed Scopus (99) Google Scholar). Several tens of thousands of peptides and thousands of proteins can be routinely identified from a single sample injection in shotgun proteomics (2Mann M. Kulak N.A. Nagaraj N. Cox J. The coming age of complete, accurate, and ubiquitous proteomes.Mol. Cell. 2013; 49: 583-590Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Shotgun proteomics, however, is limited by low analytical reproducibility. This is due to the complexity of the samples that results in under sampling (supplemental Fig. 1) and to the fact that the acquisition of MS2 spectra is often triggered outside of the elution peak apex. As a result, only 17% of the detectable peptides are typically fragmented, and less than 60% of those are identified. This translates in reliable identification of only 10% of the detectable peptides (3Michalski A. Cox J. Mann M. More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS.J. Proteome Res. 2011; 10: 1785-1793Crossref PubMed Scopus (476) Google Scholar). The overlap of peptide identification across technical replicates is typically 35–60% (4Tabb D. Vega-Montoto L. Rudnick P.A. Variyath A.M. Ham A.J. Bunk D.M. Kilpatrick L.E. Billheimer D.D. Blackman R.K. Cardasis H.L. Carr S.A. Clauser K.R. Jaffe J.D. Kowalski K.A. Neubert T.A. Regnier F.E. Schilling B. Tegeler T.J. Wang M. Wang P. Whiteaker J.R. Zimmerman L.J. Fisher S.J. Gibson B.W. Kinsinger C.R. Mesri M. Rodriguez H Stein S.E. Tempst P. Paulovich A.G. Liebler D.C. Spiegelman C. Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry.J. Proteome Res. 2009; 9: 761-776Crossref Scopus (389) Google Scholar), which results in inconsistent peptide quantification. Alternatively to shotgun proteomics, selected reaction monitoring (SRM) enables quantification of up to 200–300 peptides at very high reproducibility, accuracy, and precision (5Barnidge D.R. Dratz E.A Martin T. Bonilla L.E. Moran L.B. Lindall A. Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards.Anal. Chem. 2003; 75: 445-451Crossref PubMed Scopus (196) Google Scholar, 6Gerber S.A. Rush J. Stemman O. Kirschner M.W. Gygi S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6940-6945Crossref PubMed Scopus (1542) Google Scholar, 7Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell. Proteomics. 2007; 6: 2212-2229Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 8Gillette M.A. Carr S.A. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry.Nat. Methods. 2013; 10: 28-34Crossref PubMed Scopus (359) Google Scholar). Data-independent acquisition (DIA), a novel acquisition type, overcomes the semistochastic nature of shotgun proteomics (9Venable J. Dong M. Wohlschlegel J. Automated approach for quantitative analysis of complex peptide mixtures from tandem mass spectra.Nat. Methods. 2004; 1: 39-45Crossref PubMed Scopus (509) Google Scholar, 10Plumb R.S. Johnson K.A. Rainville P. Smith B.W. Wilson I.D. Castro-Perez J.M. Nicholson J.K. UPLC/MSE; a new approach for generating molecular fragment information for biomarker structure elucidation.Rapid Commun. Mass Spectrom. 2006; 20: 1989-1994Crossref PubMed Scopus (389) Google Scholar, 11Distler U. Kuharev J. Navarro P. Levin Y. Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics.Nat. Methods. 2015; 11Google Scholar, 12Moran D. Cross T. Brown L.M. Colligan R.M. Dunbar D. Data-independent acquisition (MSE) with ion mobility provides a systematic method for analysis of a bacteriophage structural proteome.J. Virol. Methods. 2014; 195: 9-17Crossref PubMed Scopus (16) Google Scholar, 13Geiger T. Cox J. Mann M. Proteomics on an Orbitrap benchtop mass spectrometer using all-ion fragmentation.Mol. Cell. Proteomics. 2010; 9: 2252-2261Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 14Panchaud A. Jung S. Shaffer S.A Aitchison J.D. Goodlett D.R. Faster, quantitative, and accurate precursor acquisition independent from ion count.Anal. Chem. 2011; 83: 2250-2257Crossref PubMed Scopus (65) Google Scholar, 15Pak H. Nikitin F. Gluck F. Lisacek F. Scherl A. Muller M. Clustering and filtering tandem mass spectra acquired in data-independent mode.J. Am. Soc. Mass Spectrom. 2013; 24: 1862-1871Crossref PubMed Scopus (13) Google Scholar, 16Weisbrod C.R. Eng J.K. Hoopmann M.R. Baker T. Bruce J.E. Accurate peptide fragment mass analysis: Multiplexed peptide identification and quantification.J. Proteome Res. 2012; 11: 1621-1632Crossref PubMed Scopus (72) Google Scholar, 17Carvalho P.C. Han X. Xu T. Cociorva D. Carvalho Mda. G. Barbosa V.C. Yates 3rd., J.R. XDIA: Improving on the label-free data-independent analysis.Bioinformatics. 2010; 26: 847-848Crossref PubMed Scopus (70) Google Scholar, 18Egertson J.D. Kuehn A. Merrihew G.E. Bateman N.W. MacLean B.X. Ting Y.S. Canterbury J.D. Marsh D.M. Kellmann M. Zabrouskov V. Wu C.C. MacCoss M.J. Multiplexed MS/MS for improved data-independent acquisition.Nat. Methods. 2013; 10: 744-746Crossref PubMed Scopus (207) Google Scholar). Spectra are acquired according to a predefined schema instead of dependent on the analysis of DIA data was introduced with SWATH-MS Navarro P. S. H. N. L. R. Aebersold R. data extraction of the MS/MS spectra by data-independent a new for and accurate proteome Proteomics. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). the the mass spectrometer 32 25 precursor and fragment ion spectra Navarro P. S. H. N. L. R. Aebersold R. data extraction of the MS/MS spectra by data-independent a new for and accurate proteome Proteomics. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). This results in a of detectable of the selected mass The of SWATH-MS was in the analysis of the DIA fragment are using which results in targeted are by in O. L. and L. and for of data independent acquisition of the on Mass and B. D.M. N. M. B. R. Liebler D.C. MacCoss M.J. An for and targeted proteomics 2010; 26: PubMed Scopus Google Scholar), and H.L. G. Navarro P. L. Collins J. L. Aebersold R. enables targeted analysis of data-independent acquisition 2014; PubMed Scopus Google Scholar). The of peptide identification is on the method L. O. P. Hittenhain R. M. Aebersold R. Automated data and for Methods. 2011; PubMed Scopus Google Scholar). We a novel DIA hyper reaction monitoring in in mass independent analysis and hyper reaction Rev. Proteomics. 2013; 10: PubMed Scopus Google implemented on a of DIA acquisition and targeted data analysis with retention-time-normalized spectral libraries C. L. MacLean B. R. F. J. MacCoss M.J. O. Using a retention time for targeted of 2012; PubMed Scopus Google Scholar). high of peptide identification and quantification is due to we a improved DIA we the L. O. P. Hittenhain R. M. Aebersold R. Automated data and for Methods. 2011; PubMed Scopus Google approach in the we and retention-time-normalized (iRT) spectral We compared HRM and shotgun proteomics in of to differentially abundant proteins. this we used a sample with proteins at into a stable human cell protein This in quasi complete data sets for HRM and the detection of a number of differentially abundant proteins as compared with shotgun proteomics. We HRM to identify changes in the proteome in three-dimensional human liver A. J. D. for in using and a as an 2013; PubMed Scopus Google Scholar, H. M.R. A. of proteomics of in Sci. 2011; PubMed Scopus Google Scholar, S. J.M. type human liver for 2013; PubMed Scopus Google Scholar). a of only the abundance of proteins was quantified an novel proteins adducts that might be relevant for the toxicity of were and quantified on proteins. was from and were from was from The were by and were from was from was from peptides were from of was by in and The was with for at the was with 25 for at The was to and with at a to at for The samples were at at for The peptides were using from The according to the peptides were in and of and and were as for the cell The HRM was to of the samples according to for the DIA analysis using of human and human were in with S. J.M. type human liver for 2013; PubMed Scopus Google Scholar). The liver were treated at with and in the for of were with single from were in an the were at for at and with with as The were in of and for and at for at the samples were as for the cell of the samples was on a analytical with at using an to a mass spectrometer The peptides were by a of from to with at by a to in and for the method from was used with the C. R. and acquisition for shotgun proteomics on a Orbitrap mass Proteome Res. 2012; 11: PubMed Scopus Google Scholar). The was The for the MS/MS was to collision was 10% at The HRM DIA method of a at from to of injection DIA windows were acquired at and for injection (supplemental collision was 10% at The spectra were in The MS/MS spectra were from to was on a to of on a using a as with time and width. sample was with the stable isotope The mass spectrometric data were at the the is and the is The DIA data were with a mass spectrometer from The were used for the time type was to for was to on MS2 was The false discovery rate was to at peptide The spectra were with the analysis using with the J. Mann M. enables high peptide identification mass and protein 26: PubMed Scopus Google Scholar). The peptide was to of as a of and as The mass for the precursor was and for the fragment was The were the human the in proteins and the peptide The identifications were to of on peptide and protein of the spectral measurements of the sample measurements of the were spectra were as and a spectral was using spectral in to H. Eng J.K. N. Stein S.E. Aebersold R. and of a spectral method for peptide identification from 2007; PubMed Scopus Google Scholar). from of and fragment the retention the of the assays of the a was the The was to the profiling sample set, peptides the proteins, and from were that were identified multiple using the identification was Full peptide were as peptide precursor species and The were with of and to of than was using and analysis of using 2009; PubMed Scopus Google Scholar). both and the of the peptides were across the samples and and for in a implemented in M. T. D. T. MacLean B. O. An for analysis of quantitative mass proteomic 2014; PubMed Scopus Google Scholar). The is sample precursor and the of the and the of the that are by the systematic of in the number of runs the of the of the of the the of the of The is for the of a with The was used to the of for The were for multiple using the method Y. Y. the false discovery and powerful approach to multiple R. Soc. Scholar). by the was to to The was used to the and the under the The under the with two acquisition were compared using the D.M. the under two a PubMed Scopus Google Scholar). novel acquisition method was to enable high DIA on a of and DIA that to the ion complexity of mass and This method is the new DIA of which enables of DIA windows with This the time by a of as compared with the used method for DIA R. S. O. and L. multiplexed protein profiling across sets of of acquisition with shotgun on Mass and the time of the SWATH-MS acquisition method Navarro P. S. H. N. L. R. Aebersold R. data extraction of the MS/MS spectra by data-independent a new for and accurate proteome Proteomics. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). The DIA method is to the used peak resulting on in to data of peptides from spectra of the DIA we a mass spectrometer Spectronaut. and is to L. O. P. Hittenhain R. M. Aebersold R. Automated data and for Methods. 2011; PubMed Scopus Google Scholar), with fragment can be the nature of DIA the of and for peak and time information can be used to the elution of the peptides by of This can be of as a which the of ion extraction windows with the of a of the to of peptides peptide quantification is improved by of an detection The method of is for the of the resulting J.E. Gygi S.P. for mass Mol. 2010; PubMed Scopus Google Scholar). The implemented in was by peptides in from a human cell The of the was compared with the of the spectra of the peptides the profiling of HRM and to with the established shotgun proteomics, we a of controlled mixtures the profiling sample proteins (supplemental were into a The profiling sample two for mass spectrometric detection of a number of differentially abundant proteins a of proteins and quantification. The proteins were into were to in and 60% at of detection and at and The was in a at the of detection Spectra from the profiling sample were acquired on a mass spectrometer in and DIA in technical The of runs was to and O. of quantitative mass proteomic Proteome Res. 2009; PubMed Scopus Google Scholar). the targeted analysis of the HRM we a spectral using shotgun proteomics of peptide assays for protein with an of the HRM approach can only identify and peptides and proteins for which the assays are in the spectral the content of the spectral the to the of shotgun proteomics runs at the we a of spectral libraries from an number of The was controlled at protein L. M. S.P. M. A. J.M. Aebersold R. identification false discovery for very proteomics data sets by tandem mass Cell. Proteomics. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). The number of proteins in the to to the peptides to runs of shotgun proteomics the peptide protein in the the into the the peptide identifications were for the of at fragment and for on in retention The DIA spectra were in targeted with using the spectral was The retention time extraction for the targeted HRM was by The implemented in in a of the and (supplemental Fig. peptides were identified in The spectra of the profiling sample were with the J. Mann M. enables high peptide identification mass and protein 26: PubMed Scopus Google Scholar). was peptides were identified in shotgun proteomics. the HRM approach identified on 60% peptides in a single than shotgun proteomics. This is due to the nature of precursor with DIA and to the spectral the two data was on the peptide to for and S.J. Johnson Smith for systematic with mass spectrometry and label-free proteomics Proteome Res. 2006; PubMed Scopus Google (supplemental Fig. in of identification was for HRM and shotgun proteomics. the HRM data of peptides of runs of peptides of proteins and shotgun proteomics of HRM a quasi complete data set, with only of those are compared with for shotgun proteomics This the of the targeted HRM to identify peptides and the quantitative precision of shotgun proteomics and HRM, we the of variation for the peptides that were quantified in both and detected in the The of the HRM were than of the of shotgun proteomics of for HRM, for shotgun The results for shotgun proteomics are with the which for technical replicates on the mass spectrometer (4Tabb D. Vega-Montoto L. Rudnick P.A. Variyath A.M. Ham A.J. Bunk D.M. Kilpatrick L.E. Billheimer D.D. Blackman R.K. Cardasis H.L. Carr S.A. Clauser K.R. Jaffe J.D. Kowalski K.A. Neubert T.A. Regnier F.E. Schilling B. Tegeler T.J. Wang M. Wang P. Whiteaker J.R. Zimmerman L.J. Fisher S.J. Gibson B.W. Kinsinger C.R. Mesri M. Rodriguez H Stein S.E. Tempst P. Paulovich A.G. Liebler D.C. Spiegelman C. Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry.J. Proteome Res. 2009; 9: 761-776Crossref Scopus (389) Google Scholar). the for HRM were to be than those for shotgun proteomics the (supplemental Fig. analysis of the protein that HRM and peptide for and and Fig. We quantitative of the identified peptides to the of the two to differentially abundant proteins in the profiling sample The were for shotgun proteomics and for HRM, using a of implemented in M. T. D. T. MacLean B. O. An for analysis of quantitative mass proteomic 2014; PubMed Scopus Google Scholar). protein and of the of the the was used to the The of of of mixtures were used to of proteins, by the in the for HRM and shotgun proteomics. The of proteins was for of protein and the of the of abundance was on the on the of changes in the and the proteins. Shotgun proteomics identified the number of proteins. The at the of that the HRM approach identified than as in proteins The revealed the of HRM and HRM shotgun proteomics was to changes of with the HRM a better to changes of 60% and (supplemental Fig. The of three-dimensional human liver from was used to identify relevant proteomic changes using HRM to the of on the the liver were treated with a of and the The was by an of on the was with a of to subtoxic and and were for HRM profiling. spectral was from the samples peptides of protein with on The samples were acquired in DIA using a (supplemental The spectra were with Spectronaut. peptides were identified The high reproducibility of peptide detection in a quasi complete data set, the of and technical of were from measurements and The of peptides identified in technical replicates were on The was using the of of in of proteins compared with proteins and in M.R. H. and of in to and Res. 2013; PubMed Scopus Google novel and as and were of was at the as S. E. M. to proteins and toxicity Rev. PubMed Scopus Google were which are of to M.R. H. and of in to and Res. 2013; PubMed Scopus Google Scholar). proteins for were identified at was from the and were up at The for was and the for was The of the and protein was using stable isotope (supplemental Fig. proteins, as of cell PubMed Scopus Google and protein were into which is to and to proteins. the measurements of the of was identified at the on four related proteins (GATM, PARK7, PRDX6, and VDAC2) and on and HRM analysis of the peptides that are detectable at of APAP. The peptides for the and Fig. We a of quantification of HRM and shotgun proteomics using a profiling sample The HRM outperformed shotgun proteomics in number of consistently identified in precision of and in detection of differentially abundant proteins. The and of the HRM approach for profiling is the quasi complete data that can be data is the of shotgun proteomics for quantification Smith and for label-free 2012; 13: PubMed Scopus Google Scholar). The HRM approach this The data can be using e.g. as for HRM O. R. T. S. and L. novel for protein profiling on in data implemented in Spectronaut. on Mass and for shotgun proteomics N.W. S.P. MacCoss M.J. Wu C.C. peptide identification in proteomic data-dependent Cell. Proteomics. 2013; 13: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Nagaraj N. Mann M. Accurate label-free quantification by and peptide Cell. Proteomics. 2014; 13: Full Text Full Text PDF PubMed Scopus Google Scholar). We that the of HRM to the for to better and data HRM and shotgun proteomics is the of the quantified shotgun proteomics, the quantification is on the precursor in HRM on the of the on the fragment ion is less to as of for fragment is shotgun proteomics method of the acquisition time for spectra that are used for quantification. The DIA method introduced of the acquisition time for MS2 spectra that are used for quantification. The of that quantification on the MS2 is The findings are with compared the of and of MS2 J.D. Kuehn A. Merrihew G.E. Bateman N.W. MacLean B.X. Ting Y.S. Canterbury J.D. Marsh D.M. Kellmann M. Zabrouskov V. Wu C.C. MacCoss M.J. Multiplexed MS/MS for improved data-independent acquisition.Nat. Methods. 2013; 10: 744-746Crossref PubMed Scopus (207) Google Scholar). The ion of can be and in a peptide from be quantified to on (supplemental Fig. Using HRM profiling with proteins from of were identified as the HRM protein profiling for discovery novel is that the of cell by is the of the to proteins, M.R. H. and of in to and Res. 2013; PubMed Scopus Google Scholar). than of on human proteins were We profiled on proteins. an in mitochondrial oxidative stress G. S. N. B. J. H. D. O. T. R. and mitochondrial due to of protein 2010; PubMed Scopus Google the detected at R.M. Wilson M.A. R. C. S. M.J. D. M.R. The protein is due to mitochondrial Natl. Acad. Sci. U.S.A. 2004; PubMed Scopus Google Scholar). is in the of mitochondrial in liver T. N. T. S. E. M. M.J. Fisher mitochondrial and liver in J. 2009; PubMed Scopus Google Scholar). The was to be in to oxidative stress by of into the M. T. T. A. S. T. H. of a of mitochondrial is by and by with Res. S. 2014; Scholar). has been in in oxidative stress and J.K. G. the of in Commun. 2014; PubMed Scopus Google Scholar). findings that relevant of protein were which might to cell The reproducibility and high of HRM the to mass proteomics in the shotgun proteomics but is used for the has the of spectral libraries that as for HRM targeted data The of DIA acquisition for high content discovery is a novel approach with the for that has been We of M. and M.A. for of the We M. and R. for mass spectrometric sample and with

Comprehensive, reproducible and precise analysis of large sample cohorts is one of the key objectives of quantitative proteomics. Here, we present an implementation of data-independent acquisition using its parallel acquisition nature that surpasses the limitation of serial MS2 acquisition of data-dependent acquisition on a quadrupole ultra-high field Orbitrap mass spectrometer. In deep single shot data-independent acquisition, we identified and quantified 6,383 proteins in human cell lines using 2-or-more peptides/protein and over 7100 proteins when including the 717 proteins that were identified on the basis of a single peptide sequence. 7739 proteins were identified in mouse tissues using 2-or-more peptides/protein and 8121 when including the 382 proteins that were identified based on a single peptide sequence. Missing values for proteins were within 0.3 to 2.1% and median coefficients of variation of 4.7 to 6.2% among technical triplicates. In very complex mixtures, we could quantify 10,780 proteins and 12,192 proteins when including the 1412 proteins that were identified based on a single peptide sequence. Using this optimized DIA, we investigated large-protein networks before and after the critical period for whisker experience-induced synaptic strength in the murine somatosensory cortex 1-barrel field. This work shows that parallel mass spectrometry enables proteome profiling for discovery with high coverage, reproducibility, precision and scalability.

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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Comprehensive proteome quantification is crucial for a better understanding of underlying mechanisms of diseases. Liquid chromatography mass spectrometry (LC-MS) has become the method of choice for comprehensive proteome quantification due to its power and versatility. Even though great advances have been made in recent years, full proteome coverage for complex samples remains challenging due to the high dynamic range of protein expression. Additionally, when studying disease regulatory proteins, biomarkers or potential drug targets are often low abundant, such as for instance kinases and transcription factors. Here, we show that with improvements in chromatography and data analysis the single shot proteome coverage can go beyond 10 000 proteins in human tissue. In a testis cancer study, we quantified 11 200 proteins using data independent acquisition (DIA). This depth was achieved with a false discovery rate of 1% which was experimentally validated using a two species test. We introduce the concept of hybrid libraries which combines the strength of direct searching of DIA data as well as the use of large project-specific or published DDA data sets. Remarkably deep proteome coverage is possible using hybrid libraries without the additional burden of creating a project-specific library. Within the testis cancer set, we found a large proportion of proteins in an altered expression (in total: 3351; 1453 increased in cancer). Many of these proteins could be linked to the hallmarks of cancer. For example, the complement system was downregulated which helps to evade the immune response and chromosomal replication was upregulated indicating a dysregulated cell cycle.