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Updated: May 23, 2026

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)
Published on: May 1, 2017
Enhanced peptide identification by electron transfer dissociation using an improved Mascot Percolator
James C Wright1, Mark O Collins, Lu Yu
1Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Hinxton, Cambridge.
Mascot Percolator V2.0 significantly enhances peptide identification in proteomics by improving database search software for tandem mass spectrometry, increasing identifications by up to 80% for CID and 60% for ETD fragmentation.
Area of Science:
- Proteomics
- Computational Biology
- Mass Spectrometry
Background:
- Peptide identification via tandem mass spectrometry is crucial in proteomics.
- Electron Transfer Dissociation (ETD) complements Collision Induced Dissociation (CID) for peptide fragmentation.
- Optimal database search software has been a limitation for ETD efficacy.
Purpose of the Study:
- To report developments in Mascot Percolator V2.0 software.
- To evaluate the software's performance with CID and ETD fragmented peptide data.
- To assess the complementarity of CID and ETD fragmentation techniques.
Main Methods:
- Developed Mascot Percolator V2.0 with an improved feature calculator and wider ion series support.
- Applied the updated software to analyze CID and ETD fragmented peptide datasets.
- Utilized a dataset of CID and ETcaD spectral pairs for comparative analysis.
Main Results:
- Mascot Percolator V2.0 increased CID PSMs by up to 80% and ETD PSMs by up to 60% at a 1% FDR.
- The software recovered PSMs from high charge state precursor ions.
- Overlap in peptide identifications between CID and ETD was 83% using Mascot Percolator, higher than standalone Mascot (69%) or OMSSA (39%).
Conclusions:
- Mascot Percolator V2.0 is a highly sensitive and accurate post-search algorithm for peptide identification.
- The software enables direct comparison of peptide identifications across multiple fragmentation techniques.
- Enhanced peptide identification and coverage facilitate a fuller assessment of CID/ETD complementarity.
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