Abstract
A major goal of proteomics research is the accurate and sensitive identification and quantification of a broad range of proteins within a sample. Data-independent acquisition (DIA) approaches that acquire MS/MS spectra independently of precursor information have been developed to overcome the reproducibility challenges of data-dependent acquisition and the limited breadth of targeted proteomics strategies. Typical DIA implementations use wide MS/MS isolation windows to acquire comprehensive fragment ion data. However, wide isolation windows produce highly chimeric spectra, limiting the achievable sensitivity and accuracy of quantification and identification. Here, we present a DIA strategy in which spectra are collected with overlapping (rather than adjacent or random) windows and then computationally demultiplexed. This approach improves precursor selectivity by nearly a factor of 2, without incurring any loss in mass range, mass resolution, chromatographic resolution, scan speed, or other key acquisition parameters. We demonstrate a 64% improvement in sensitivity and a 17% improvement in peptides detected in a 6-protein bovine mix spiked into a yeast background. To confirm the method’s applicability to a realistic biological experiment, we also analyze the regulation of the proteasome in yeast grown in rapamycin and show that DIA experiments with overlapping windows can help elucidate its adaptation toward the degradation of oxidatively damaged proteins. Our integrated computational and experimental DIA strategy is compatible with any DIA-capable instrument. The computational demultiplexing algorithm required to analyze the data has been made available as part of the open-source proteomics software tools Skyline and msconvert (Proteowizard), making it easy to apply as part of standard proteomics workflows.Graphical
Highlights
Proteomics techniques for peptide detection and quantification in a complex sample typically begin with a tryptic digest of the sample of interest followed by liquid chromatography tandem mass spectrometry (LC-MS/MS)
The standard 10 m/z method serves as a positive control and is intended to represent the level of precursor selectivity that could be theoretically achieved if the overlapping of 20 m/z windows were fully successful in removing half of the interference in each window
The workflow is applicable to any DIAcapable instrument and can be implemented immediately with the tools required for analysis already available as part of the Skyline software package
Summary
Proteomics techniques for peptide detection and quantification in a complex sample typically begin with a tryptic digest of the sample of interest followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometry (MS/MS) is almost always necessary for the unambiguous confirmation of the peptide sequence in a. A Bsurvey^ MS scan is performed; peptide precursors of interest (usually the most intense ions) are selected for fragmentation (MS/MS analysis) to determine their amino acid sequence. DDA is designed for Bdiscovery-based^ analysis, where the goal is to detect a broad range of peptides present in the sample, rather than to measure specific peptides of interest. The only signal that is measured systematically is the MS1 precursor which is limited in selectivity and, in ion trapping instruments, the MS1 signal from a spectrum spanning a large m/z range is limited in dynamic range because of space charge limits
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Journal of the American Society for Mass Spectrometry
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
