NDLI: Identification of the Unpaired Cysteine Status and Complete Mapping the 17 Disulfides of Recombinant Tissue Plasminogen Activator Using LC-MS with ETD/CID

Content Provider	PubMed Central
Author	Wu, Shiaw Lin Jiang, Haitao Hancock, William S. Karger, Barry L.
Abstract	Recombinant tissue plasminogen (rt-PA) with 35 cysteine residues has been completely assigned by mapping the 17 disulfide linkages and the unpaired cysteine. The result is consistent with the prediction from homology except for the unassigned cysteine, which was identified at Cys83. This cysteine was found to be blocked and paired with either a glutathione or cysteine residue in a ~ 60 :40 ratio, respectively. The analysis was conducted using a multi-fragmentation approach consisting of ETD and CID, in combination with a multi-enzyme digestion strategy (Lys-C, trypsin, and Glu-C). The disulfide-linked peptides, even those containing N or O-linked glycosylation, could be assigned since the disulfide bonds were still preferably cleaved over the glycosidic cleavages under ETD fragmentation. The use of a multiple and sequential enzymatic digestion strategy was important in producing fragment sizes suitable for analysis. For the analysis of complex intertwined disulfides, the use of CID MS3 to target partially disulfide dissociated peptides from the ETD fragmentation was necessary for linkage assignment. The ability to identify the exact location and status of the unpaired cysteine (free or blocked with a glutathione or cysteine) could shed light on the activation of rt-PA, upon stimulation by either oxidative or ischemic stress.
Related Links	http://dx.doi.org/10.1021/ac100766r
Ending Page	5303
Page Count	8
Starting Page	5296
File Format	PDF
ISSN	00032700
e-ISSN	15206882
Journal	Analytical chemistry
Issue Number	12
Volume Number	82
Language	English
Publisher Date	2010-06-15
Access Restriction	Open
Subject Keyword	Analytical Chemistry Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Analytical Chemistry

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