TY - JOUR
T1 - Continuous Elution Proteoform Identification of Myelin Basic Protein by Superficially Porous Reversed-Phase Liquid Chromatography and Fourier Transform Mass Spectrometry
AU - Plymire, Daniel A.
AU - Wing, Casey E.
AU - Robinson, Dana E.
AU - Patrie, Steven M.
N1 - Funding Information:
S.M.P. contributed to project concept, oversight, software/ code, data tabulation, and writing. D.E.R. and C.E.W. contributed to software and data analysis. D.A.P. contributed to data analysis and writing. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number 1R01GM115739-01A1. Any opinions, findings, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Institutes of Health. This work was also supported by the Multiple Sclerosis Society [PP-1503-04034], The Darrel K. Royal Research Fund for Alzheimer’s Disease [48680-DKR], The Texas Alzheimer’s Research and Care Consortium Investigator Grant Program [354091], and the UT System Neuroscience and Neuro-technology Research Institute [363027], and The John L. Roach Scholarship in Biomedical Research.
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/11/21
Y1 - 2017/11/21
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.analchem.7b02426
DO - 10.1021/acs.analchem.7b02426
M3 - Article
C2 - 29016107
AN - SCOPUS:85034739508
SN - 0003-2700
VL - 89
SP - 12030
EP - 12038
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 22
ER -
