| 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: Publisher |
Abstract/OtherAbstract:
|
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. |
| | |
Authors:
|
Wenqin Ni; Melanie Lin; Paul Salinas; Philip Savickas; Shiaw-Lin Wu; Barry L Karger |
Related Documents
:
|
11000005 - Development of peptide 3d structure mimetics: rational design of novel peptoid cholecys... 18509495 - History and diagnostic significance of c-peptide. 18812635 - Role of feed-regulating peptides on pancreatic exocrine secretion. 7991445 - Distribution of gastrin-releasing peptide/bombesin-like immunoreactivity in the rainbow... 794835 - Structlre of transfer rna molecules containing the long variable loop. 9677335 - Amino acid sequence of glutathione s-transferase rgstm5* from rat testis. |
Publication Detail:
|
Type: JOURNAL ARTICLE Date: 2012-12-4 |
Journal Detail:
|
Title: Journal of the American Society for Mass Spectrometry Volume: - ISSN: 1879-1123 ISO Abbreviation: J. Am. Soc. Mass Spectrom. Publication Date: 2012 Dec |
Date Detail:
|
Created Date: 2012-12-4 Completed Date: - Revised Date: - |
Medline Journal Info:
|
Nlm Unique ID: 9010412 Medline TA: J Am Soc Mass Spectrom Country: - |
Other Details:
|
Languages: ENG Pagination: - Citation Subset: - |
Affiliation:
|
Barnett Institute of Chemical and Biological Analysis and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, 02115, USA. |
Export Citation:
|
APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
|
|
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
Previous Document: Gas-Phase Reactivity of Carboxylic Acid Functional Groups with Carbodiimides.
Next Document: Delayed visual recovery in pregnancy-associated thrombotic thrombocytopenic purpura with bilateral s...