Quantitative analysis of discovery-based proteomic work- flows 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. Molecular & Cellular Proteomics 14: 10.1074/mcp.O115.048181, 2015.

Response Curves for a set of Acetylated Peptides in a Complex Matrix—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). Mouse Liver Mitochondrial Protein Lysate—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. PHDE1α Kinase Inhibitor Study—Mouse liver mitochondria from wild-type mice (C57BL/6) were isolated as described previously (29, 30). 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). Heavy phosphopeptides for pSer-293 (YHGHS293MSDPGVSYR[13C615N4]) and pSer-300 (YHGHSMS300DPGVSYR[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.

Data sets acquired on a TripleTOF 5600 mass spectrometer: 1) MS1 and SWATH-MS – Kinase Inhibitor Study for quantifying changes in endogenous phosphopeptide isomers YHGHpS293MSDPGVSYR and YHGHSMSDPGVpS300YR from PDHE1a (3 injection replicates), go to Kinase Inhibitor Study in Panorama, download Kinase Inhibitor Study. 2) MS1 and SWATH-MS – dilution curve of heavy labeled phosphopeptide isomers YHGHpS293MSDPGVSYR and YHGHSMSDPGVpS300YR from PDHE1a (3 injection replicates), download various isomer ratio sub files: 16:1/1:16, 8:1/1:8, 4:1/1:4, 1:2, 1:1. 3) MS1 and SWATH-MS – Optimization of SWATH window size (25 and 10 m/z, respectively) to reduce interferences in a mitochondrial lysate (5 injection replicates), go to SWATH Optimization Study in Panorama, download SWATH Optimization Study. 4) MS1 and SWATH-MS – MS1 and MS2 Data Processing Study for removal of interferences using mitochondrial lysates at a concentration of 100 ng or 33 ng (2 process replicates, 3 injection replicates), go to Data Processing Study in Panorama, download Data Processing Study (sample legend).