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Related Concept Videos

Peptide Identification Using Tandem Mass Spectrometry01:33

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Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
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A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
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Updated: Oct 1, 2025

Navigating the Mass Spectrometry-Based Proteomic Data Using Free Computational Tools
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Data-Independent Acquisition Protease-Multiplexing Enables Increased Proteome Sequence Coverage Across Multiple

Alicia L Richards1,2,3, Kuei-Ho Chen1,2,3, Damien B Wilburn4,5,6

  • 1Quantitative Biosciences Institute (QBI), University of California San Francisco, San Francisco, California 94158, United States.

Journal of Proteome Research
|March 2, 2022
PubMed
Summary

Multiplexing proteases in data-independent acquisition (DIA) proteomics increases peptide detections and amino acid coverage. This approach enhances protein analysis, particularly for identifying noncanonical protein isoforms.

Keywords:
CIDDIA-MSisoformslabel-free quantificationmultiplexingproteases

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Biochemistry

Background:

  • Multiple proteases improve protein sequence coverage in proteomics but are limited by analysis time in data-dependent acquisition (DDA).
  • Data-independent acquisition (DIA) offers potential for multiplexed samples but is optimized for tryptic peptides.

Purpose of the Study:

  • To evaluate a DIA multiplexing approach combining three proteolytic digests (Trypsin, AspN, GluC) in a single sample.
  • To optimize data acquisition conditions for multiplexed protease digests in DIA.
  • To assess the impact on protein identification, quantification, and sequence coverage.

Main Methods:

  • Optimization of DIA acquisition conditions for individual proteases (Trypsin, AspN, GluC) using canonical (beam type CID) and resonance excitation CID fragmentation.
  • Application of optimized conditions to protease-multiplexed human peptide samples.
  • Analysis of protein identifications, peptide detections, amino acid detections, and sequence coverage.

Main Results:

  • Optimized consensus conditions were determined for multiplexed protease analysis in DIA.
  • The multiplexing approach yielded similar protein identifications and quantitative performance compared to trypsin alone.
  • A significant increase was observed in peptide detections (up to 63%) and nonredundant amino acid detections (45%).
  • Nontryptic peptides facilitated noncanonical protein isoform determination and achieved 100% sequence coverage for several proteins.

Conclusions:

  • DIA multiplexing with multiple proteases is a viable strategy to enhance peptide and amino acid detection in proteomics.
  • This method significantly improves sequence coverage, enabling critical applications like protein isoform analysis.
  • The approach offers a powerful tool for comprehensive proteomic profiling and isoform characterization.