Continuous Elution Proteoform Identification of Myelin Basic Protein by Superficially Porous Reversed-Phase Liquid Chromatography and Fourier Transform Mass Spectrometry

Abstract

Myelin basic protein (MBP) plays an important structural and functional role in the neuronal myelin sheath. Translated MBP exhibits extreme microheterogeneity with numerous alternative splice variants (ASVs) and post-translational modifications (PTMs) reportedly tied to central nervous system maturation, myelin stability, and the pathobiology of various de- and dys-myelinating disorders. Conventional bioanalytical tools cannot efficiently examine ASV and PTM events simultaneously, which limits understanding of the role of MBP microheterogeneity in human physiology and disease. To address this need, we report on a top-down proteomics pipeline that combines superficially porous reversed-phase liquid chromatography (SPLC), Fourier transform mass spectrometry (FTMS), data-independent acquisition (DIA) with nozzle-skimmer dissociation (NSD), and aligned data processing resources to rapidly characterize abundant MBP proteoforms within murine tissue. The three-tier proteoform identification and characterization workflow resolved four known MBP ASVs and hundreds of differentially modified states from a single 90 min SPLC-FTMS run on ~0.5 μg of material. This included 323 proteoforms for the 14.1 kDa ASV alone. We also identified two novel ASVs from an alternative transcriptional start site (ATSS) of the MBP gene as well as a never before characterized S-acylation event linking palmitic acid, oleic acid, and stearic acid at C78 of the 17.125 kDa ASV.

Top-Down and Intact Protein Mass Spectrometry Data Visualization for Proteoform Analysis Using VisioProt-MS

“…In addition, directly comparing these maps reveals differences in proteoform footprints between samples and/or experimental conditions. 1,8,[12][13][14][15][16] Recently, we developed a free standalone tool to facilitate this analytical step: VisioProt-MS. 17 Here, we will illustrate its use through the analysis of the 20S core particle of the human proteasome.…”

Exploring bioactive peptides from bacterial secretomes using PepSAVI‐MS: identification and characterization of Bac‐21 from Enterococcus faecalis pPD1

“…While providing a wealth of information to use for peptide sequencing, interpretation of these MS/MS spectra is often complex and confident fragment assignment is often ambiguous in many cases. Automatic software programs are crucial to facilitate this process, and an in‐house custom informatics search engine (Plymire et al ., ) was used in this case. As such, the accurate intact mass of Bac‐21 was determined to be 7145.071 ‐0 Da (7145.072 ‐0 Da theoretical; 0.3 ppm mass accuracy) with 70% sequence coverage obtained via CID fragmentation of the +6 charge state (Fig.…”

“…Data analysis was accomplished using a custom informatics search engine adapted from Plymire et al . (). The candidate sequences were tested against the fragmentation data at 15 ppm mass tolerance.…”

“…CID fragmentation was performed on the +6 Bac-21 charge state (1192 m/z) with a collisional energy of 35 V and 600-2000 m/z range. Data analysis was accomplished using a custom informatics search engine adapted from Plymire et al (2017). The candidate sequences were tested against the fragmentation data at 15 ppm mass tolerance.…”

VisioProt-MS: interactive 2D maps from intact protein mass spectrometry

“…However, only a handful include LC-MS 2 D maps produced with in-house tools. These include publications from the groups of Kelleher (Durbin et al , 2010; Tran et al , 2011), Patrie (Plymire et al , 2017), and Smith (Lee et al , 2002). We published several papers presenting such 2 D maps using RoWinPro deconvolution (Gersch et al , 2015) plotted with Excel (Carel et al , 2017; Parra et al , 2017), but this workflow is very low throughput and does not facilitate dynamic data inspection.…”