MS1 Peptide Ion Intensity Chromatograms in MS2 (SWATH) Data Independent Acquisitions. Improving Post Acquisition Analysis of Proteomic Experiments

Quantitative analysis of discovery-based proteomic workflows now relies on high-throughput large-scale methods for identification and quantitation of proteins and post-translational modifications. Advancements in label-free quantitative techniques, using either data-dependent or data-independent mass spectrometric acquisitions, have coincided with improved instrumentation featuring greater precision, increased mass accuracy, and faster scan speeds. We recently reported on a new quantitative method called MS1 Filtering (Schilling et al. (2012) Mol. Cell. Proteomics 11, 202–214) for processing data-independent MS1 ion intensity chromatograms from peptide analytes using the Skyline software platform. In contrast, data-independent acquisitions from MS2 scans, or SWATH, can quantify all fragment ion intensities when reference spectra are available. As each SWATH acquisition cycle typically contains an MS1 scan, these two independent label-free quantitative approaches can be acquired in a single experiment. Here, we have expanded the capability of Skyline to extract both MS1 and MS2 ion intensity chromatograms from a single SWATH data-independent acquisition in an Integrated Dual Scan Analysis approach. The performance of both MS1 and MS2 data was examined in simple and complex samples using standard concentration curves. Cases of interferences in MS1 and MS2 ion intensity data were assessed, as were the differentiation and quantitation of phosphopeptide isomers in MS2 scan data. In addition, we demonstrated an approach for optimization of SWATH m/z window sizes to reduce interferences using MS1 scans as a guide. Finally, a correlation analysis was performed on both MS1 and MS2 ion intensity data obtained from SWATH acquisitions on a complex mixture using a linear model that automatically removes signals containing interferences. This work demonstrates the practical advantages of properly acquiring and processing MS1 precursor data in addition to MS2 fragment ion intensity data in a data-independent acquisition (SWATH), and provides an approach to simultaneously obtain independent measurements of relative peptide abundance from a single experiment. Quantitative analysis of discovery-based proteomic workflows now relies on high-throughput large-scale methods for identification and quantitation of proteins and post-translational modifications. Advancements in label-free quantitative techniques, using either data-dependent or data-independent mass spectrometric acquisitions, have coincided with improved instrumentation featuring greater precision, increased mass accuracy, and faster scan speeds. We recently reported on a new quantitative method called MS1 Filtering (Schilling et al. (2012) Mol. Cell. Proteomics 11, 202–214) for processing data-independent MS1 ion intensity chromatograms from peptide analytes using the Skyline software platform. In contrast, data-independent acquisitions from MS2 scans, or SWATH, can quantify all fragment ion intensities when reference spectra are available. As each SWATH acquisition cycle typically contains an MS1 scan, these two independent label-free quantitative approaches can be acquired in a single experiment. Here, we have expanded the capability of Skyline to extract both MS1 and MS2 ion intensity chromatograms from a single SWATH data-independent acquisition in an Integrated Dual Scan Analysis approach. The performance of both MS1 and MS2 data was examined in simple and complex samples using standard concentration curves. Cases of interferences in MS1 and MS2 ion intensity data were assessed, as were the differentiation and quantitation of phosphopeptide isomers in MS2 scan data. In addition, we demonstrated an approach for optimization of SWATH m/z window sizes to reduce interferences using MS1 scans as a guide. Finally, a correlation analysis was performed on both MS1 and MS2 ion intensity data obtained from SWATH acquisitions on a complex mixture using a linear model that automatically removes signals containing interferences. This work demonstrates the practical advantages of properly acquiring and processing MS1 precursor data in addition to MS2 fragment ion intensity data in a data-independent acquisition (SWATH), and provides an approach to simultaneously obtain independent measurements of relative peptide abundance from a single experiment. Mass spectrometry is the leading technology for large-scale identification and quantitation of proteins and post-translational modifications (PTMs) 1 in biological systems (1.Olsen J.V. Mann M. Status of large-scale analysis of post-translational modifications by mass spectrometry.Mol. Cell. Proteomics. 2013; 12: 3444-3452Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 2.Bantscheff M. Lemeer S. Savitski M.M. Kuster B. Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present.Anal. Bioanal. Chem. 2012; 404: 939-965Crossref PubMed Scopus (581) Google Scholar). Although several types of experimental designs are employed in such workflows, most large-scale applications use data-dependent acquisitions (DDA) where peptide precursors are first identified in the MS1 scan and one or more peaks are then selected for subsequent fragmentation to generate their corresponding MS2 spectra. In experiments using DDA, one can employ either chemical/metabolic labeling or label-free strategies for relative quantitation of peptides (and proteins) (3.Neilson K.A. Ali N.A. Muralidharan S. Mirzaei M. Mariani M. Assadourian G. Lee A. van Sluyter S.C. Haynes P.A. Less label, more free: approaches in label-free quantitative mass spectrometry.Proteomics. 2011; 11: 535-553Crossref PubMed Scopus (537) Google Scholar, 4.Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar). Depending on the type of labeling approach employed, i.e. metabolic labeling with SILAC or postmetabolic labeling with ICAT or isobaric tags such as iTRAQ or TMT, the relative quantitation of these peptides are made using either MS1 or MS2 ion intensity data (4.Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar, 5.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar, 6.Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4341) Google Scholar, 7.Thompson A. Schafer J. Kuhn K. Kienle S. Schwarz J. Schmidt G. Neumann T. Johnstone R. Mohammed A.K. Hamon C. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.Anal. Chem. 2003; 75: 1895-1904Crossref PubMed Scopus (1709) Google Scholar). Label-free quantitative techniques have until recently been based entirely on integrated ion intensity measurements of precursors in the MS1 scan, or in the case of spectral counting the number of assigned MS2 spectra (3.Neilson K.A. Ali N.A. Muralidharan S. Mirzaei M. Mariani M. Assadourian G. Lee A. van Sluyter S.C. Haynes P.A. Less label, more free: approaches in label-free quantitative mass spectrometry.Proteomics. 2011; 11: 535-553Crossref PubMed Scopus (537) Google Scholar, 8.Ishihama Y. Oda Y. Tabata T. Sato T. Nagasu T. Rappsilber J. Mann M. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.Mol. Cell. Proteomics. 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In a a MS1 scan is the mass by a SWATH MS2 acquisition the cycle of the MS1 scan is with type The is to a typically to the cell in the mass the spectra a SWATH MS2 acquisition are of greater as the spectra are a of all fragment from peptide analytes with the selected MS1 m/z The cycle of data independent MS1 scans and SWATH MS2 scans is the ion in these SWATH MS2 spectra can be to peptides by to reference spectra or spectral ion intensities of these fragment can be for Although MS2 typically and the in MS1 scans, peptides from SWATH MS2 scans can be of the of interferences in one or more fragment or ion intensity of MS2 scans as with the MS1 precursor ion of these in SWATH MS2 scan quantitation is to MS1 ion intensity is acquired as of each SWATH scan and have quantitation for data acquisitions B. D.J. and label-free quantitation of proteomic data using MS1 ion chromatograms in to protein and Cell. Proteomics. 2012; 11: Full Text Full Text PDF PubMed Scopus Google Scholar, J. I. N. 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S. as a of to Full Text Full Text PDF PubMed Scopus Google Scholar). peptides based on MS1 precursor ion intensities can be by a This is the case when abundance peptides in a complex where the MS1 ion is or when chromatograms or of precursor MS1 scans are or by we an that data-independent acquisitions experiments by MS1 ion intensity data in the data processing of to we have Skyline to extract and both precursor and ion chromatograms for quantitation The and for and MS1 Filtering experiments have been expanded to data from and expanded a single for data of proteomics using both the MS1 and MS2 scans that we Integrated Dual Scan or As a of a of SWATH MS2 acquisitions of simple and complex mixtures was on an mass We the use of MS2 scans for a case of phosphopeptide isomers that are the MS1 In addition, we SWATH m/z more quantitative data in these by the number of interferences. we performed a of the and of the of from concentration to the of acquiring MS1 and MS2 data in and as independent measurements and were obtained from for protein and were from protein were was from and were from Proteomics was from was from peptides containing and were to generate standard concentration in either a simple protein or a complex on from to concentration and for the and concentration each with from to concentration were acquired on the MS2 were by from and proteins were with and were then with for 1 with in the and with a were then with and using were to and to were in and a concentration of or were acquired in a on the mass either as is or an of for from were as S.E. 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S. H. N. R. Aebersold R. data of the spectra by data-independent a new for and accurate Cell. Proteomics. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). As quantitation in is using ion intensities of MS2 the of MS1 precursors in these workflows have been the that their ion are In case a MS1 scan of of the acquisition for the of We have a new approach as of the Skyline software for of ion intensity measurements from MS1 and MS2 scans, called Integrated Dual Scan Analysis that several both MS1 and MS2 data can be and from a single acquisition in or we that data can be to MS2 ion for peptide and be for of