SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries

Abstract

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. Data Independent Acquisition (DIA) 1 mass spectrometry workflows are gaining increasing use for proteomic analysis of model systems (1-8). The first integrated DIA and quantitative analysis protocol, termed SWATH (2) was shown to offer accurate, reproducible, and robust proteomic quantification (9 -14). DIA offers advantages over conventional IDA methods (15) by overcoming the stochastic, intensity-based selection of peptide precursors-a problem which typically leads to inconsistent peptide detection and quantitation between replicate runs. By overcoming this problem, DIA is highly suited for large-scale comparative analyses as gaps in data points between samples are mostly eliminated. These digital, extensive proteome maps can be repeatedly mined for quantitative data by extracting ion chromatograms of defined peptides postacquisition, and yields fewer quantitative missing (NA) values than IDA. An important concept in DIA analysis is use of a LC-retention time referenced spectral ion assay library to enable peptide identification from DIA generated multiplexed MS/MS spectra (10,13,16). The depth and quality of this spectral reference library directly correlates with experimental outcome, therefore we consider it is essential to explore and understand this variable in detail.The reference assay library should contain all the prior knowle...

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

“…Immunopurified Kal7 samples were cleaved using in-gel tryptic digestion (V51111; Promega, Madison WI) and subjected to information-dependent acquisition and SWATH acquisition, as described 36, 84-86 . Skyline (v2.4) 87 was employed for precursor ion quantification using SWATH data for the various phosphorylation sites.…”

Identification of small molecules using accurate mass MS/MS search

“…There are a number of R‐language computational packages hosted on BioConductor (http://www.bioconductor.org/) that can be used to manipulate and process MS/MS spectra. That includes MSnID, MSnbase, msPurity, RMassBank, SwathXtend, and RAMClustR …”

Insights into the study and origin of the citrullinome in rheumatoid arthritis

“…The first advantage of the approach is a more comprehensive fragmentation, including low abundance sub‐proteomes, such as citrullination. The second advantage is the reference assay library that should contain all the prior knowledge of the peptide components to be extracted from the SWATH data . Thus, assay library generation is one key challenge and limitation of this approach.…”