Development of a Top-Down Proteomics Platform for Characterizing Myelin Basic Protein Proteoforms
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A top-down proteomic analysis platform for the analysis of myelin basic protein was designed and applied to murine central nervous system tissue. The compatibility of superficially porous (SP) resin for label-free intact protein analysis of myelin basic protein with on-line capillary liquid chromatography mass spectrometry was demonstrated and was able to resolve splice variants and individual proteoforms of myelin basic protein. Data independent acquisition using nozzle skimmer dissociation yielded fragment ions that were used with novel informatics procedures to identify proteoforms of MBP. The informatics procedures including continuous elution proteoform scoring, a three-tier search methodology, and a ranking system for identification validation yielded 323 unique proteoforms from a single splice variant of MBP from a single LC run. A novel site of acylation and two novel splice variants derived from a novel transcriptional start site were also identified.