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Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human
Michael J Roth1, Andrew J Forbes, Michael T Boyne
1Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, Illinois 61801, USA.
Molecular & Cellular Proteomics : MCP
|May 3, 2005
Summary
This study introduces a new database and top-down mass spectrometry (MS/MS) approach for characterizing human proteins and their variants. This method precisely identifies genetic variations and modifications, advancing proteome annotation.
Area of Science:
- Proteomics
- Genomics
- Biochemistry
Background:
- The human proteome's complexity, arising from alternative splicing, RNA editing, and posttranslational modifications, challenges comprehensive protein characterization.
- High sequence identity within gene families further complicates the routine analysis of mammalian proteins using mass spectrometry (MS).
Purpose of the Study:
- To develop and apply a novel database and top-down MS/MS strategy for the comprehensive characterization of the human proteome.
- To demonstrate the capability of this approach for identifying gene-specific proteins and characterizing diverse protein variants and modifications.
Main Methods:
- Creation of a comprehensive human protein database including potential variants.
- Application of top-down tandem mass spectrometry (MS/MS) to analyze intact proteins from HeLa cells.
- Utilizing precise mass shift (Deltam) measurements for variant and modification identification.
Main Results:
- Successful gene-specific identification of 45 distinct protein forms from HeLa cell proteome.
- Characterization of 34 coding single nucleotide polymorphisms, 2 alternative splicing variants, and 12 diverse posttranslational modifications.
- Discovery of a previously unidentified phosphorylation event at 10% occupancy.
- Automated protein identification achieved with a median expectation value of 10(-13).
Conclusions:
- Top-down MS/MS is a powerful tool for the precise annotation of gene products within the complex human proteome.
- This approach enables simultaneous identification of proteins and dissection of their inherent variability, including genetic and posttranslational modifications.
- The developed method holds significant promise for broader adoption by researchers, including non-mass spectrometrists, for detailed proteomic analysis.