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Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot
Published on: October 28, 2021
Sequence scrambling in shotgun proteomics is negligible
Anton A Goloborodko1, Mikhail V Gorshkov, David M Good
1Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia.
Journal of the American Society for Mass Spectrometry
|September 29, 2011
Summary
Sequence scrambling in mass spectrometry is negligible for peptide analysis. However, non-scrambled internal fragments in higher-energy collisional dissociation (HCD) require consideration by MS/MS search engines.
Area of Science:
- Proteomics
- Mass Spectrometry
- Analytical Chemistry
Background:
- Shotgun proteomics relies on tandem mass spectrometry (MS/MS) for peptide identification.
- Accurate peptide sequencing is crucial for reliable proteome analysis.
- Collisional dissociation methods like Collision-Induced Dissociation (CID) and Higher-Energy Collisional Dissodciation (HCD) are used to generate fragment ions.
Purpose of the Study:
- To investigate the extent of peptide sequence scrambling caused by b-ion cyclization during low-energy (CID) and higher-energy (HCD) collisional dissociation.
- To assess the impact of sequence scrambling on the accuracy of peptide sequence assignment in shotgun proteomics.
- To identify and characterize the presence of normal internal fragments in HCD spectra.
Main Methods:
- Analysis of a large dataset comprising 15,897 CID and 10,878 HCD mass spectra.
- High-resolution mass spectrometry was employed for detailed spectral analysis.
- Evaluation of b-ion cyclization and the formation of internal fragments.
Main Results:
- The rate of sequence scrambling due to b-ion cyclization was found to be negligible (<1%) in both CID and HCD.
- This low rate of scrambling indicates it can be safely ignored as a source of erroneous sequence assignment.
- Significant presence of normal (non-scrambled) internal fragments was observed in HCD spectra.
Conclusions:
- Peptide sequence scrambling is not a significant issue in high-resolution MS/MS analysis of tryptic peptides.
- The findings support the reliability of current peptide sequencing methods in shotgun proteomics.
- MS/MS search engines should incorporate the detection of normal internal fragments in HCD spectra for improved accuracy.
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