Abstract
Proteomics by mass spectrometry technology is widely used for identifying and quantifying peptides and proteins. The breadth and sensitivity of peptide detection have been advanced by the advent of data-independent acquisition mass spectrometry. Analysis of such data, however, is challenging due to the complexity of fragment ion spectra that have contributions from multiple co-eluting precursor ions. We present SWATHProphet software that identifies and quantifies peptide fragment ion traces in data-independent acquisition data, provides accurate probabilities to ensure results are correct, and automatically detects and removes contributions to quantitation originating from interfering precursor ions. Integration in the widely used open source Trans-Proteomic Pipeline facilitates subsequent analyses such as combining results of multiple data sets together for improved discrimination using iProphet and inferring sample proteins using ProteinProphet. This novel development should greatly help make data-independent acquisition mass spectrometry accessible to large numbers of users.
Highlights
Lower intensity, in samples [2]
SWATHProphet software was designed to analyze data-independent acquisition (DIA) MS data with precursor ion assays of a spectral library
Combining SWATHProphet Results of Multiple Runs and Protein Inference—Accurate SWATHProphet probabilities can serve as input to downstream analyses in the highly developed Trans-Proteomic Pipeline (TPP) that apply both to DIA and shotgun MS results
Summary
Automated Validation of Results and Removal of Fragment Ion Interferences in Targeted Analysis of Data-independent Acquisition Mass Spectrometry (MS) using SWATHProphet*□S. This novel development should greatly help make data-independent acquisition mass spectrometry accessible to large numbers of users. The proprietary Spectronaut (Biognosys AG, Zurich, Switzerland) and open source OpenSWATH software [9] are adaptations of the mProphet software suite [10] originally designed for SRM data, and the widely used SRM software Skyline [11] incorporates mProphet software to handle DIA MS data None of these available programs, provide validation of results with computed probabilities or detection and removal of fragment ion interferences that give rise to inaccurate quantitation and decreased sensitivity. SWATHProphet uses these probabilities to help cope with complex spectra by automatically detecting fragment ion interferences and removing them in silico to yield accurate quantitation and adjusted probabilities
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