Accurate Identification of Deamidation and Citrullination from Global Shotgun Proteomics Data Using a Dual-Search Delta Score Strategy
While proteins with deamidated/citrullinated amino acids play critical roles in the pathogenesis of many human diseases, identifying these modifications in complex biological samples has been an ongoing challenge. Herein we present a method to accurately identify these modifications from shotgun proteomics data from a deep proteome profiling study of human pancreatic islets obtained by laser capture microdissection. All MS/MS spectra were searched against database by MSGF+ twice with or without a +0.9840 Da mass shift on amino acids asparagine, glutamine, and arginine (NQR) as a dynamic modification. Consequently, for each spectrum the resulting two peptide-to-spectrum matches (PSM) with their respective MSGF+ scores were used for Delta Score calculation. It was observed that all PSMs with positive Delta Score values were clustered with mass errors around 0 ppm, while PSMs with negative Delta Score values were distributed nearly equally within the defined mass error range (20 ppm) for database searching. To estimate false discovery rate (FDR), a “pseudo-decoy” approach was applied whether datasets were searched against a database with a “real modification” mass shift (+0.9840 Da) and a “mock modification” mass shift (+1.0227 Da). FDR was controlled to ~2% with a Delta Score filter greater than zero. Manual inspection of spectra showed that PSMs with positive Delta Score value contained deamidated/citrullinated fragments in their MS/MS spectra. Finally, the results demonstrated that in-situ deamidated/citrullinated peptides can be accurately identified from shotgun tissue proteomics data by the Dual-search Delta Score Strategy.
- Research Organization:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE; National Institutes of Health (NIH); China Scholarship Council (CSC)
- Grant/Contract Number:
- AC05-76RL01830; DP3 DK110844; R01 DK122160; P41GM103493
- OSTI ID:
- 1618832
- Report Number(s):
- PNNL-SA-149222
- Journal Information:
- Journal of Proteome Research, Vol. 19, Issue 4; ISSN 1535-3893
- Publisher:
- American Chemical Society (ACS)Copyright Statement
- Country of Publication:
- United States
- Language:
- English
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