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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
Effect of 2MEGA labeling on membrane proteome analysis using LC-ESI QTOF MS
Chengjie Ji1, Andy Lo, Sandra Marcus
1Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Journal of Proteome Research
|October 7, 2006
Summary
2MEGA labeling enhances proteome analysis by improving peptide fragmentation and reducing false positives in mass spectrometry. This method increases identified proteins, particularly membrane proteins, in Escherichia coli.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Large-scale proteome analysis via tandem mass spectrometry (MS/MS) faces challenges in minimizing false positive identifications.
- Automated database searching requires strategies to balance true positive discovery with reduced false positives.
Purpose of the Study:
- To investigate the impact of 2MEGA labeling on proteome analysis of Escherichia coli membrane fractions using 2D LC-MS/MS.
- To assess the effect of 2MEGA labeling on peptide fragmentation patterns and false positive rates.
Main Methods:
- Systematic comparison of MS/MS spectra from native and 2MEGA-labeled peptides.
- Proteome analysis of Escherichia coli membrane fraction using 2D LC-MS/MS.
- Identification of proteins using MASCOT database search algorithm.
Main Results:
- 2MEGA labeling facilitates facile peptide fragmentation, enhancing detection of N-terminal a1-related ions.
- The method alleviates biased detection of arginine-terminated peptides.
- 2MEGA labeling increased the number of identified peptides and proteins, including a significant rise in integral membrane proteins (153 vs. 77).
Conclusions:
- 2MEGA labeling improves proteome coverage and accuracy in MS/MS analysis.
- Enhanced a1-related ion generation serves as a constraint to reduce false positive identifications.
- This technique is particularly effective for identifying membrane proteins in complex biological samples.
