Document Detail

Complete mapping of a cystine knot and nested disulfides of recombinant human arylsulfatase A by multi-enzyme digestion and LC-MS analysis using CID and ETD.
MedLine Citation:
PMID:  23208745     Owner:  NLM     Status:  MEDLINE    
Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS(3) (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138-Cys154, Cys143-Cys150, Cys282-Cys396, Cys470-Cys482, Cys471-Cys484, and Cys475-Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70%). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.
Wenqin Ni; Melanie Lin; Paul Salinas; Philip Savickas; Shiaw-Lin Wu; Barry L Karger
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-12-04
Journal Detail:
Title:  Journal of the American Society for Mass Spectrometry     Volume:  24     ISSN:  1879-1123     ISO Abbreviation:  J. Am. Soc. Mass Spectrom.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-24     Completed Date:  2013-07-01     Revised Date:  2014-01-09    
Medline Journal Info:
Nlm Unique ID:  9010412     Medline TA:  J Am Soc Mass Spectrom     Country:  United States    
Other Details:
Languages:  eng     Pagination:  125-33     Citation Subset:  IM    
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MeSH Terms
Amino Acid Sequence
Cerebroside-Sulfatase / chemistry*,  metabolism
Chromatography, Liquid / methods
Cysteine / chemistry*,  metabolism
Disulfides / chemistry*,  metabolism
Mass Spectrometry / methods
Molecular Sequence Data
Peptide Fragments / analysis,  chemistry*,  metabolism
Peptide Hydrolases / metabolism
Peptide Mapping / methods*
Recombinant Proteins / chemistry,  metabolism
Grant Support
Reg. No./Substance:
0/Disulfides; 0/Peptide Fragments; 0/Recombinant Proteins; EC; EC 3.4.-/Peptide Hydrolases; K848JZ4886/Cysteine

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