MS1 Peptide Ion Intensity Chromatograms in MS2 (SWATH) Data Independent Acquisitions. Improving Post Acquisition Analysis of Proteomic Experiments
2015; Elsevier BV; Volume: 14; Issue: 9 Linguagem: Inglês
10.1074/mcp.o115.048181
ISSN1535-9484
AutoresMatthew J. Rardin, Birgit Schilling, Lin-Yang Cheng, Brendan MacLean, Dylan J. Sorensen, Alexandria K. Sahu, Michael J. MacCoss, Olga Vitek, Bradford W. Gibson,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoQuantitative 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. 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Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.Mol. Cell. Proteomics. 2012; 11: 202-214Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 12.Cox J. Hein M.Y. Luber C.A. Paron I. Nagaraj N. Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.Mol. Cell. Proteomics. 2014; 13: 2513-2526Abstract Full Text Full Text PDF PubMed Scopus (2687) Google Scholar), in part because of their adaptability to a wide range of proteomic workflows, including human samples that are not amenable to most metabolic labeling techniques, or where chemical labeling may be cost prohibitive and/or interfere with subsequent enrichment steps (11.Schilling B. Rardin M.J. MacLean B.X. Zawadzka A.M. Frewen B.E. Cusack M.P. Sorensen D.J. Bereman M.S. Jing E. Wu C.C. Verdin E. Kahn C.R. Maccoss M.J. Gibson B.W. Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.Mol. Cell. Proteomics. 2012; 11: 202-214Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 13.Qian W.J. Jacobs J.M. Liu T. Camp 2nd, D.G. Smith R.D. Advances and challenges in liquid chromatography-mass spectrometry-based proteomics profiling for clinical applications.Mol. Cell. Proteomics. 2006; 5: 1727-1744Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). However the use of DDA for label-free quantitation is also susceptible to several limitations including insufficient reproducibility because of under-sampling, digestion efficiency, as well as misidentifications (14.Tabb D.L. 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. 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Shotgun collision-induced dissociation of peptides using a time of flight mass analyzer.Proteomics. 2003; 3: 847-850Crossref PubMed Scopus (130) Google Scholar). Shortly thereafter Venable et al. reported on a data independent acquisition methodology to limit the complexity of the MS2 scan by using a segmented approach for the sequential isolation and fragmentation of all peptides in a defined precursor window (e.g. 10 m/z) using an ion trap mass spectrometer (17.Venable J.D. Dong M.Q. Wohlschlegel J. Dillin A. Yates J.R. Automated approach for quantitative analysis of complex peptide mixtures from tandem mass spectra.Nat. Methods. 2004; 1: 39-45Crossref PubMed Scopus (509) Google Scholar). However, the proper implementation of this DIA technique suffered from technical limitations of instruments available at that time, including slow acquisition rates and low MS2 resolution that made systematic product ion extraction problematic. To alleviate the challenge of long duty cycles in DIAs, researchers at the Waters Corporation adopted an alternative approach by rapidly switching between low (MS1) and high energy (MS2) scans and then using proprietary software to align peptide precursor and fragment ion information to determine peptide sequences (18.Silva J.C. Denny R. Dorschel C.A. Gorenstein M. Kass I.J. Li G.Z. McKenna T. Nold M.J. Richardson K. Young P. Geromanos S. Quantitative proteomic analysis by accurate mass retention time pairs.Anal. Chem. 2005; 77: 2187-2200Crossref PubMed Scopus (521) Google Scholar, 19.Silva J.C. Gorenstein M.V. Li G.Z. Vissers J.P. Geromanos S.J. Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition.Mol. Cell. Proteomics. 2006; 5: 144-156Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar). 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Multiplexed MS/MS for improved data-independent acquisition.Nat. Methods. 2013; 10: 744-746Crossref PubMed Scopus (207) Google Scholar). Moreover, the simultaneous development of novel software solutions for extracting ion intensity chromatograms based on spectral libraries has enabled the use of DIA for large-scale label free quantitation of multiple peptide analytes (21.Egertson 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, 22.Rost H.L. Rosenberger G. Navarro P. Gillet L. Miladinovic S.M. Schubert O.T. Wolski W. Collins B.C. Malmstrom J. Malmstrom L. Aebersold R. OpenSWATH enables automated, targeted analysis of data-independent acquisition MS data.Nat. Biotechnol. 2014; 32: 219-223Crossref PubMed Scopus (500) Google Scholar). In addition to targeting specific peptides from a previously generated peptide spectral library, the data can also be reexamined (i.e. post-acquisition) for additional peptides of interest as new reference data emerges. On the SCIEX TripleTOF 5600, a quadrupole orthogonal time-of-flight mass spectrometer, this technique has been optimized and extended to what is called 'SWATH MS2′ based on a combination of new technical and software improvements (10.Gillet L.C. Navarro P. Tate S. Rost H. Selevsek N. Reiter L. Bonner R. Aebersold R. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis.Mol. Cell. Proteomics. 2012; 11O111.016717Abstract Full Text Full Text PDF PubMed Scopus (1777) Google Scholar, 22.Rost H.L. Rosenberger G. Navarro P. Gillet L. Miladinovic S.M. Schubert O.T. Wolski W. Collins B.C. Malmstrom J. Malmstrom L. Aebersold R. OpenSWATH enables automated, targeted analysis of data-independent acquisition MS data.Nat. Biotechnol. 2014; 32: 219-223Crossref PubMed Scopus (500) Google Scholar). In a DIA experiment a MS1 survey scan is carried out across the mass range followed by a SWATH MS2 acquisition series, however the cycle time of the MS1 scan is dramatically shortened compared with DDA type experiments. The Q1 quadrupole is set to transmit a wider window, typically Δ25 m/z, to the collision cell in incremental steps over the full mass range. Therefore the MS/MS spectra produced during a SWATH MS2 acquisition are of much greater complexity as the MS/MS spectra are a composite of all fragment ions produced from peptide analytes with molecular ions within the selected MS1 m/z window. The cycle of data independent MS1 survey scans and SWATH MS2 scans is repeated throughout the entire LC-MS acquisition. Fragment ion information contained in these SWATH MS2 spectra can be used to uniquely identify specific peptides by comparisons to reference spectra or spectral libraries. Moreover, ion intensities of these fragment ions can also be used for quantitation. Although MS2 typically increases selectivity and reduces the chemical noise often observed in MS1 scans, quantifying peptides from SWATH MS2 scans can be problematic because of the presence of interferences in one or more fragment ions or decreased ion intensity of MS2 scans as compared with the MS1 precursor ion abundance. To partially alleviate some of these limitations in SWATH MS2 scan quantitation it is potentially advantageous to exploit MS1 ion intensity data, which is acquired independently as part of each SWATH scan cycle. Recently, our laboratories and others have developed label free quantitation tools for data dependent acquisitions (11.Schilling B. Rardin M.J. MacLean B.X. Zawadzka A.M. Frewen B.E. Cusack M.P. Sorensen D.J. Bereman M.S. Jing E. Wu C.C. Verdin E. Kahn C.R. Maccoss M.J. Gibson B.W. Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.Mol. Cell. Proteomics. 2012; 11: 202-214Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 12.Cox J. Hein M.Y. Luber C.A. Paron I. Nagaraj N. Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.Mol. Cell. Proteomics. 2014; 13: 2513-2526Abstract Full Text Full Text PDF PubMed Scopus (2687) Google Scholar, 23.Sandin M. Teleman J. Malmstrom J. Levander F. Data processing methods and quality control strategies for label-free LC-MS protein quantification.Biochim. Biophys. Acta. 2014; 1844: 29-41Crossref PubMed Scopus (46) Google Scholar) using MS1 ion intensity data. For example, the MS1 Filtering algorithm uses expanded features in the open source software application Skyline (11.Schilling B. Rardin M.J. MacLean B.X. Zawadzka A.M. Frewen B.E. Cusack M.P. Sorensen D.J. Bereman M.S. Jing E. Wu C.C. Verdin E. Kahn C.R. Maccoss M.J. Gibson B.W. Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.Mol. Cell. Proteomics. 2012; 11: 202-214Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 24.MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2963) Google Scholar). Skyline MS1 Filtering processes precursor ion intensity chromatograms of peptide analytes from full scan mass spectral data acquired during data dependent acquisitions by LC MS/MS. New graphical tools were developed within Skyline to enable visual inspection and manual interrogation and integration of extracted ion chromatograms across multiple acquisitions. MS1 Filtering was subsequently shown to have excellent linear response across several orders of magnitude with limits of detection in the low attomole range (11.Schilling B. Rardin M.J. MacLean B.X. Zawadzka A.M. Frewen B.E. Cusack M.P. Sorensen D.J. Bereman M.S. Jing E. Wu C.C. Verdin E. Kahn C.R. Maccoss M.J. Gibson B.W. Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.Mol. Cell. Proteomics. 2012; 11: 202-214Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). We, and others, have demonstrated the utility of this method for carrying out large-scale quantitation of peptide analytes across a range of applications (25.Guo A. Gu H. Zhou J. Mulhern D. Wang Y. Lee K.A. Yang V. Aguiar M. Kornhauser J. Jia X. Ren J. Beausoleil S.A. Silva J.C. Vemulapalli V. Bedford M.T. Comb M.J. Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.Mol. Cell. Proteomics. 2014; 13: 372-387Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 26.Rardin M.J. He W. Nishida Y. Newman J.C. Carrico C. Danielson S.R. Guo A. Gut P. Sahu A.K. Li B. Uppala R. Fitch M. Riiff T. Zhu L. Zhou J. Mulhern D. Stevens R.D. Ilkayeva O.R. Newgard C.B. Jacobson M.P. Hellerstein M. Goetzman E.S. Gibson B.W. Verdin E. SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks.Cell Metab. 2013; 18: 920-933Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 27.Rardin M.J. Newman J.C. Held J.M. Cusack M.P. Sorensen D.J. Li B. Schilling B. Mooney S.D. Kahn C.R. Verdin E. Gibson B.W. Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 6601-6606Crossref PubMed Scopus (342) Google Scholar, 28.Sos M.L. Levin R.S. Gordan J.D. Oses-Prieto J.A. Webber J.T. Salt M. Hann B. Burlingame A.L. McCormick F. Bandyopadhyay S. Shokat K.M. Oncogene mimicry as a mechanism of primary resistance to BRAF inhibitors.Cell Reports. 2014; 8: 1037-1048Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, quantifying peptides based on MS1 precursor ion intensities can be compromised by a low signal-to-noise ratio. This is particularly the case when quantifying low abundance peptides in a complex sample where the MS1 ion "background" signal is high, or when chromatograms contain interferences, or partial overlap of multiple target precursor ions. Currently MS1 scans are underutilized or even deemphasized by some vendors during DIA workflows. However, we believe an opportunity exists that would improve data-independent acquisitions (DIA) experiments by including MS1 ion intensity data in the final data processing of LC-MS/MS acquisitions. Therefore, to address this possibility, we have adapted Skyline to efficiently extract and process both precursor and product ion chromatograms for label free quantitation across multiple samples. The graphical tools and features originally developed for SRM and MS1 Filtering experiments have been expanded to process DIA data sets from multiple vendors including SCIEX, Thermo, Waters, Bruker, and Agilent. These expanded features provide a single platform for data mining of targeted proteomics using both the MS1 and MS2 scans that we call Integrated Dual Scan Analysis, or IDSA. As a test of this approach, a series of SWATH MS2 acquisitions of simple and complex mixtures was analyzed on an SCIEX TripleTOF 5600 mass spectrometer. We also investigated the use of MS2 scans for differentiating a case of phosphopeptide isomers that are indistinguishable at the MS1 level. In addition, we investigated whether smaller SWATH m/z windows would provide more reliable quantitative data in these cases by reducing the number of potential interferences. Lastly, we performed a statistical assessment of the accuracy and reproducibility of the estimated (log) fold change of mitochondrial lysates from mouse liver at different concentration levels to better assess the overall value of acquiring MS1 and MS2 data in combination and as independent measurements during DIA experiments. HPLC solvents including acetonitrile and water were obtained from Burdick & Jackson (Muskegon, MI). Reagents for protein chemistry including iodoacetamide, dithiothreitol (DTT), ammonium bicarbonate, formic acid, trifluoroacetic acid, acetic acid, dichloroacetic acid (DCA), dodecyl-maltoside, and urea were purchased from Sigma Aldrich (St. Louis, MO). All protein standards were >95% purity. Tris(2-carboxyethyl)phosphine (TCEP) was purchased from Thermo (Rockford, IL), and HLB Oasis SPE cartridges were purchased from Waters (Milford, MA). Proteomics grade trypsin was from Promega (Madison WI). Trypsin-predigested beta-galactosidase (a quality control standard) was purchased from SCIEX (Foster City, CA). Six lysine-acetylated synthetic peptides containing 13C615N2-Lys and 13C615N4-Arg were used to generate standard concentration curves in either a simple (25 fmol "six protein mix") or a complex matrix (complex mitochondrial lysate, 0.3 μg on column), spanning from 4 attomoles to 25 femtomoles over 6 concentration points (0.004, 0.012, 0.037, 0.111, 0.333, 1, 3, and 25 fmol) for the following peptides: LVSSVSDLPKacR (HMGCS2 protein), MVQKacSLAR (HMGCS2 protein), AFVDSCLQLHETKacR (LCAD protein), YAPVAKacDLASR (SDHA protein), LFVDKacIR (ATP5J protein), and AFGGQSLKacFGK (SDHA protein). Three replicate concentration curves, each with injections from lowest to highest spike concentration were acquired on the TripleTOF 5600 (SWATH MS2 mode). Mitochondria were isolated by differential centrifugation from liver WT (C57BL/6) mice, and proteins were denatured with 1% dodecyl-maltoside and 10 m urea. Samples were then diluted 1:10, reduced with 4.5 mm TCEP (37 °C for 1 h), alkylated with 10 mm iodoacetamide (30 min at RT in the dark), and incubated overnight at 37 °C with sequencing grade trypsin added at a 1:50 enzyme/substrate ratio (wt/wt). Samples were then acidified with formic acid and desalted using HLB Oasis SPE cartridges. Samples were eluted, concentrated to near dryness, and resuspended prior to analysis. Samples were processed in duplicates and three injection replicates at a concentration of 100 ng or 33 ng were acquired in a randomized order on the TripleTOF 5600 mass spectrometer either as is or spiked into an E. coli hydrolysate of 300 ng for additional complexity. Mouse liver mitochondria from wild-type mice (C57BL/6) were isolated as described previously (29.Rardin M.J. Wiley S.E. Murphy A.N. Pagliarini D.J. Dixon J.E. Dual specificity phosphatases 18 and 21 target to opposing sides of the mitochondrial inner membrane.J. Biol. Chem. 2008; 283: 15440-15450Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 30.Rardin M.J. Wiley S.E. Naviaux R.K. Murphy A.N. Dixon J.E. Monitoring phosphorylation of the pyruvate dehydrogenase complex.Anal. Biochem. 2009; 389: 157-164Crossref PubMed Scopus (104) Google Scholar). Mitochondria (1 mg) were incubated at room temperature with 5 mm DCA (10 mm Hepes pH 7.2) for 0, 5, 10, 30, 60, and 120 min. Samples were digested with trypsin and phosphopeptides were enriched by IMAC chromatography as previously described (11.Schilling B. Rardin M.J. MacLean B.X. Zawadzka A.M. Frewen B.E. Cusack M.P. Sorensen D.J. Bereman M.S. Jing E. Wu C.C. Verdin E. Kahn C.R. Maccoss M.J. Gibson B.W. Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.Mol. Cell. Proteomics. 2012; 11: 202-214Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Heavy phosphopeptides for pSer-293 (YHGHS293MSDPGVSYR[13C615N4]) and pSer-300 (YHGHSMSDPGVS300YR[13C615N4]) were spiked at 25 fmol into each sample. Equal volumes of eluted phosphopeptides were desalted using C-18 zip-tips and then analyzed on the TripleTOF 5600. Mass spectrometric data was acqui
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