Human Proteomic Variation Revealed by Combining RNA-Seq Proteogenomics and Global Post-Translational Modification (G-PTM) Search Strategy

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.