Proteolysis of multiple myelin basic protein isoforms after neurotrauma: characterization by mass spectrometry.
Neurotrauma, as in the case of traumatic brain injury, promotes protease over-activation characterized by the select fragmentation of brain proteins. The resulting polypeptides are indicators of biochemical processes, which can be used to study post-injury dynamics and may also be developed into biomarkers. To this end, we devised a novel mass spectrometry approach to characterize post-injury calpain proteolytic processing of myelin basic protein (MBP), a biomarker of brain injury that denotes white matter damage and recovery. Our approach exceeds conventional immunological assays in its deconvolution of multiple protein isoforms, its absolute quantification of proteolytic fragments and its polypeptide selectivity. We quantified and characterized post-injury proteolytic processing of all MBP isoforms identified in adult rat cortex. Further, the translation of calpain-cleaved MBP into CSF was verified following brain injury. We ascertained that the exon-6 sequence of MBP resulted in a characteristic shift in gel migration for intact and fragmented protein alike. We also found evidence for a second post-TBI cleavage event within exon-2 and for the dimerization of the post-TBI 4.3 kDa fragment. Ultimately, the novel methodology described here can be used to study MBP dynamics and other similar proteolytic events of relevance to brain injury and other CNS processes.
Department of Psychiatry, Centers for Neuroproteomics and Biomarker Research and Traumatic Brain Injury Studies at the McKnight Brain Institute of the University of Florida, Gainesville, Florida 32610-0256, USA. aottens@mbi.ufl.edu
Andrew K
AK
Ottens
Erin C
EC
Golden
Liliana
L
Bustamante
Ronald L
RL
Hayes
Nancy D
ND
Denslow
Kevin K W
KK
Wang
D000595:Amino Acid Sequence; D000818:Animals; D001930:Brain Injuries / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006868:Hydrolysis; D008297:Male; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry / Q000379:methods; D013997:Time Factors
D004676:Myelin Basic Protein; D020033:Protein Isoforms; D002154:Calpain; D003546:Cysteine Endopeptidases; C019913:clostripain