Splice-Junction-Based Mapping of Alternative Isoforms in the Human Proteome

Abstract

Summary: The protein-level translational status and function of many alternative splicing events remain poorly understood. We use an RNA sequencing (RNA-seq)-guided proteomics method to identify protein alternative splicing isoforms in the human proteome by constructing tissue-specific protein databases that prioritize transcript splice junction pairs with high translational potential. Using the custom databases to reanalyze ∼80 million mass spectra in public proteomics datasets, we identify more than 1,500 noncanonical protein isoforms across 12 human tissues, including ∼400 sequences undocumented on TrEMBL and RefSeq databases. We apply the method to original quantitative mass spectrometry experiments and observe widespread isoform regulation during human induced pluripotent stem cell cardiomyocyte differentiation. On a proteome scale, alternative isoform regions overlap frequently with disordered sequences and post-translational modification sites, suggesting that alternative splicing may regulate protein function through modulating intrinsically disordered regions. The described approach may help elucidate functional consequences of alternative splicing and expand the scope of proteomics investigations in various systems. : The translation and function of many alternative splicing events await confirmation at the protein level. Lau et al. use an integrated proteotranscriptomics approach to identify non-canonical and undocumented isoforms from 12 organs in the human proteome. Alternative isoforms interfere with functional sequence features and are differentially regulated during iPSC cardiomyocyte differentiation. Keywords: alternative splicing, splice isoforms, protein isoforms, proteoforms, mass spectrometry, proteomics, induced pluripotent stem cells, intrinsically disordered region, human proteome, cardiomyocyte differentiation