Advances in Mass Spectrometry Instrumentation and Methodology for Analysis of Alternative Protein Isoforms

Proteoforms represent the many final protein products that can be generated by a protein-coding gene. A key source of proteomic variation is alternative splicing (AS), with at least 90% of human genes undergoing AS to cause protein sequence alterations that impact protein structure and function. Therefore, detection and characterization of the protein isoforms generated by splicing is critical to a complete understanding of the effects of splicing on human phenotype and disease. Advancements in the power and throughput of mass spectrometry (MS) instrumentation over the past decade, coupled with growing interest in splicing among the proteomics community, are driving a new wave of MS utilization for protein isoform analysis. In this review, we outline recent innovations in MS instruments and the new acquisition strategies they support, sample preparation protocols, and integrative bioinformatics pipelines that have enabled deeper sampling of the alternative proteome. We highlight research from the field utilizing bottom-up and top-down MS as well as discovery and targeted acquisition methods, outlining the isoform coverage and advantages offered by each approach. Based on the evolution of the field, splicing continues to garner greater attention within the context of proteoform diversity.