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J Proteome Res. 2014 Jan 3;13(1):228-40. doi: 10.1021/pr4009207. Epub 2013 Nov 11.

Large-scale mass spectrometric detection of variant peptides resulting from nonsynonymous nucleotide differences.

Author information

1
Department of Chemistry, University of Wisconsin-Madison , 1101 University Avenue, Madison, Wisconsin 53706, United States.

Abstract

Each individual carries thousands of nonsynonymous single nucleotide variants (nsSNVs) in their genome, each corresponding to a single amino acid polymorphism (SAP) in the encoded proteins. It is important to be able to directly detect and quantify these variations at the protein level to study post-transcriptional regulation, differential allelic expression, and other important biological processes. However, such variant peptides are not generally detected in standard proteomic analyses due to their absence from the generic databases that are employed for mass spectrometry searching. Here we extend previous work that demonstrated the use of customized SAP databases constructed from sample-matched RNA-Seq data. We collected deep-coverage RNA-Seq data from the Jurkat cell line, compiled the set of nsSNVs that are expressed, used this information to construct a customized SAP database, and searched it against deep-coverage shotgun MS data obtained from the same sample. This approach enabled the detection of 421 SAP peptides mapping to 395 nsSNVs. We compared these peptides to peptides identified from a large generic search database containing all known nsSNVs (dbSNP) and found that more than 70% of the SAP peptides from this dbSNP-derived search were not supported by the RNA-Seq data and thus are likely false positives. Next, we increased the SAP coverage from the RNA-Seq derived database by utilizing multiple protease digestions, thereby increasing variant detection to 695 SAP peptides mapping to 504 nsSNV sites. These detected SAP peptides corresponded to moderate to high abundance transcripts (30+ transcripts per million, TPM). The SAP peptides included 192 allelic pairs; the relative expression levels of the two alleles were evaluated for 51 of those pairs and were found to be comparable in all cases.

PMID:
24175627
PMCID:
PMC3947302
DOI:
10.1021/pr4009207
[Indexed for MEDLINE]
Free PMC Article

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