title: Human Proteomic Variation Revealed by Combining RNA-Seq Proteogenomics and Global Post-Translational Modification (G-PTM) Search Strategy.
abstract: Mass-spectrometry-based proteomic analysis underestimates proteomic variation due to the absence of variant peptides and posttranslational modifications (PTMs) from standard protein databases. Each individual carries thousands of missense mutations that lead to single amino acid variants, but these are missed because they are absent from generic proteomic search databases. Myriad types of protein PTMs play essential roles in biological processes but remain undetected because of increased false discovery rates in variable modification searches. We address these two fundamental shortcomings of bottom-up proteomics with two recently developed software tools. The first consists of workflows in Galaxy that mine RNA sequencing data to generate sample-specific databases containing variant peptides and products of alternative splicing events. The second tool applies a new strategy that alters the variable modification approach to consider only curated PTMs at specific positions, thereby avoiding the combinatorial explosion that traditionally leads to high false discovery rates. Using RNA-sequencing-derived databases with this Global Post-Translational Modification (G-PTM) search strategy revealed hundreds of single amino acid variant peptides, tens of novel splice junction peptides, and several hundred posttranslationally modified peptides in each of ten human cell lines.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Cesnik
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Shortreed
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Gloria M
authors.initials: GM
authors.lastname: Sheynkman
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Frey
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , 1101 University Avenue, Madison, Wisconsin 53706, United States.|Genome Center of Wisconsin, University of Wisconsin-Madison , 425G Henry Mall, Madison, Wisconsin 53706, United States.
authors.forename: Lloyd M
authors.initials: LM
authors.lastname: Smith
mesh_terms: D001483:Base Sequence / Q000235:genetics*; D002460:Cell Line; D030562:Databases, Protein; D006801:Humans; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D000071696:Proteogenomics / Q000379:methods; D040901:Proteomics / Q000379:methods*; D012984:Software
keywords: G-PTM; PTM; RNA-Seq; bottom-up proteomics; cancer cell lines; novel splice junction (NSJ); proteogenomics; proteomic database search; single amino acid variant (SAV)