Abstract
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.
Highlights
From the ‡Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, California 94945; §Department of Statistics, Purdue University, West Lafayette, IN 47907; ¶College of Science, College of Computer and Information Science, Northeastern University, Boston, Massachusetts 02115; ʈDepartment of Genome Sciences, University of Washington School of Medicine, Seattle, Washington; **Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143
MS1 and MS2 Crosstalk in Data Independent Acquisitions 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 selected for subsequent fragmentation to generate their corresponding MS2 spectra
MS Acquisition and Processing of Data Independent MS1 and MS2 Scans in Skyline—MS1 Filtering in Skyline [11] is typically employed to process full scan ion intensity chromatograms from data dependent acquisitions (Fig. 1A)
Summary
Materials—HPLC solvents including acetonitrile and water were obtained from Burdick & Jackson (Muskegon, MI). 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. SWATH MS2 data sets are targeted DIA assays, and Skyline quantitation was based on XICs of up to 10 MS/MS fragment ions, typically y- and b-ions, matching to specific peptides present in the spectral libraries described above. Data was acquired on the TripleTOF 5600 from 2 process and 3 injection replicates and analyzed as follows Because both MS1 and MS2 scans are subject to quantitative interferences and noise, we determined an initial representative quantitative profile of each peptide based on area under the curve (AUC) of XIC peaks. The MS1 MS2 SWATH data sets and Skyline files uploaded to Panorama can be accessed at https://panoramaweb.org/labkey/MS1MS2.url
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