Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase.
In this work, we explore the potential of the metalloendopeptidase Lys-N for MALDI-MS/MS proteomics applications. Initially we digested a HEK293 cellular lysate with Lys-N and, for comparison, in parallel with the protease Lys-C. The resulting peptides were separated by strong cation exchange to enr...
| Main Authors: | , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
2009
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| _version_ | 1797082354503647232 |
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| author | Boersema, P Taouatas, N Altelaar, A Gouw, J Ross, P Pappin, D Heck, A Mohammed, S |
| author_facet | Boersema, P Taouatas, N Altelaar, A Gouw, J Ross, P Pappin, D Heck, A Mohammed, S |
| author_sort | Boersema, P |
| collection | OXFORD |
| description | In this work, we explore the potential of the metalloendopeptidase Lys-N for MALDI-MS/MS proteomics applications. Initially we digested a HEK293 cellular lysate with Lys-N and, for comparison, in parallel with the protease Lys-C. The resulting peptides were separated by strong cation exchange to enrich and isolate peptides containing a single N-terminal lysine. MALDI-MS/MS analysis of these peptides yielded CID spectra with clear and often complete sequence ladders of b-ions. To test the applicability for de novo sequencing we next separated an ostrich muscle tissue protein lysate by one-dimensional SDS-PAGE. A protein band at 42 kDa was in-gel digested with Lys-N. Relatively straightforward sequencing resulted in the de novo identification of the two ostrich proteins creatine kinase and actin. We therefore conclude that this method that combines Lys-N, strong cation exchange enrichment, and MALDI-MS/MS analysis provides a valuable alternative proteomics strategy. |
| first_indexed | 2024-03-07T01:27:00Z |
| format | Journal article |
| id | oxford-uuid:92517464-bbb2-4a58-a708-a4088fbdc0c6 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T01:27:00Z |
| publishDate | 2009 |
| record_format | dspace |
| spelling | oxford-uuid:92517464-bbb2-4a58-a708-a4088fbdc0c62022-03-26T23:24:36ZStraightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:92517464-bbb2-4a58-a708-a4088fbdc0c6EnglishSymplectic Elements at Oxford2009Boersema, PTaouatas, NAltelaar, AGouw, JRoss, PPappin, DHeck, AMohammed, SIn this work, we explore the potential of the metalloendopeptidase Lys-N for MALDI-MS/MS proteomics applications. Initially we digested a HEK293 cellular lysate with Lys-N and, for comparison, in parallel with the protease Lys-C. The resulting peptides were separated by strong cation exchange to enrich and isolate peptides containing a single N-terminal lysine. MALDI-MS/MS analysis of these peptides yielded CID spectra with clear and often complete sequence ladders of b-ions. To test the applicability for de novo sequencing we next separated an ostrich muscle tissue protein lysate by one-dimensional SDS-PAGE. A protein band at 42 kDa was in-gel digested with Lys-N. Relatively straightforward sequencing resulted in the de novo identification of the two ostrich proteins creatine kinase and actin. We therefore conclude that this method that combines Lys-N, strong cation exchange enrichment, and MALDI-MS/MS analysis provides a valuable alternative proteomics strategy. |
| spellingShingle | Boersema, P Taouatas, N Altelaar, A Gouw, J Ross, P Pappin, D Heck, A Mohammed, S Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase. |
| title | Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase. |
| title_full | Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase. |
| title_fullStr | Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase. |
| title_full_unstemmed | Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase. |
| title_short | Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase. |
| title_sort | straightforward and de novo peptide sequencing by maldi ms ms using a lys n metalloendopeptidase |
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