Fast Quantitative Analysis of timsTOF PASEF Data with MSFragger and IonQuant
Fengchao Yu1, Sarah E Haynes1, Guo Ci Teo1
1Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.
Molecular & Cellular Proteomics : MCP
|July 4, 2020
Summary
New software, MSFragger and IonQuant, accelerates the analysis of timsTOF PASEF mass spectrometry data, identifying more peptides and proteins faster than existing tools. This advancement enables more comprehensive proteomic studies and discovery of post-translational modifications.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Ion mobility separation coupled with mass spectrometry (LC-MS) enhances peptide and protein identification in complex biological samples.
- The timsTOF mass spectrometer utilizes trapped ion mobility spectrometry (TIMS) and parallel accumulation serial fragmentation (PASEF) for advanced proteomic analysis.
- Current data analysis tools for timsTOF PASEF data, such as MaxQuant and PEAKS, are time-consuming and limit complex search strategies.
Purpose of the Study:
- To develop and validate faster and more comprehensive software tools for analyzing timsTOF PASEF data.
- To improve peptide and protein identification rates and quantification accuracy for complex proteomic samples.
- To enable advanced search strategies, including semi-enzymatic and open searches, for enhanced proteome coverage.
Main Methods:
- Extension of the MSFragger tool to support timsTOF PASEF data processing.
- Development of the IonQuant tool for label-free quantification and 4-D feature extraction.
- Validation using a public HeLa dataset and comparison with MaxQuant and PEAKS X+.
- Performance evaluation based on peptide/protein identification rates, quantification accuracy, and processing runtime.
Main Results:
- MSFragger identified approximately 30% more unique peptides than MaxQuant and comparable or better results than PEAKS X+.
- IonQuant achieved superior protein quantification compared to existing tools, with good precision and accuracy.
- MSFragger and IonQuant processed a 2-hour PASEF run in under 70 minutes on standard hardware.
- Semi-enzymatic searching significantly increased peptide identification, revealing evidence of pre-MS/MS gas-phase fragmentation.
Conclusions:
- MSFragger and IonQuant provide a significantly faster and more comprehensive solution for timsTOF PASEF data analysis.
- The developed tools overcome current bottlenecks, enabling advanced proteomic investigations and PTM discovery.
- The findings demonstrate the potential for increased proteome coverage and novel biological insights using these enhanced analytical methods.
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