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. 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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). 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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). 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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. 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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. 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