Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions.
Trypsin, the most widely used enzyme in proteomics, has a few caveats as it does not perform well under certain harsh sample handling conditions and creates relatively short peptides less amenable to, for instance, electron transfer dissociation. There is, thus, room for improvement using alternativ...
| Main Authors: | , , |
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| Format: | Journal article |
| Language: | English |
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2010
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| _version_ | 1826286680199397376 |
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| author | Taouatas, N Heck, A Mohammed, S |
| author_facet | Taouatas, N Heck, A Mohammed, S |
| author_sort | Taouatas, N |
| collection | OXFORD |
| description | Trypsin, the most widely used enzyme in proteomics, has a few caveats as it does not perform well under certain harsh sample handling conditions and creates relatively short peptides less amenable to, for instance, electron transfer dissociation. There is, thus, room for improvement using alternative proteases. Here, we evaluate the performance of such an alternative protease, the metalloendopeptidase Lys-N, in sample preparation for proteomic analyses under various experimental conditions. The experimental parameters we evaluated were protein-to-protease ratio, incubation time, temperature, and several concentrations of denaturing modifiers often used in proteomics sample handling. Our data reveal that Lys-N is still very efficient under some very harsh (denaturing) conditions (e.g., 8 M urea, 80% acetonitrile) and at temperatures as low as 4 degrees C and up to 80 degrees C but severely hampered by guanidine hydrochloride and methanol. These rather unique features make Lys-N a good candidate for a variety of applications, such as membrane proteomics and possibly H/D exchange mass spectrometry. Additionally, we show that Lys-N is capable of, in contrast to trypsin or Lys-C, cleaving adjacent to mono- and dimethylated lysines, making it a good candidate for targeted epigenetic analysis of for instance histones. |
| first_indexed | 2024-03-07T01:47:20Z |
| format | Journal article |
| id | oxford-uuid:98e0c16d-2210-4228-9406-2bd8e66c6516 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T01:47:20Z |
| publishDate | 2010 |
| record_format | dspace |
| spelling | oxford-uuid:98e0c16d-2210-4228-9406-2bd8e66c65162022-03-27T00:10:02ZEvaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:98e0c16d-2210-4228-9406-2bd8e66c6516EnglishSymplectic Elements at Oxford2010Taouatas, NHeck, AMohammed, STrypsin, the most widely used enzyme in proteomics, has a few caveats as it does not perform well under certain harsh sample handling conditions and creates relatively short peptides less amenable to, for instance, electron transfer dissociation. There is, thus, room for improvement using alternative proteases. Here, we evaluate the performance of such an alternative protease, the metalloendopeptidase Lys-N, in sample preparation for proteomic analyses under various experimental conditions. The experimental parameters we evaluated were protein-to-protease ratio, incubation time, temperature, and several concentrations of denaturing modifiers often used in proteomics sample handling. Our data reveal that Lys-N is still very efficient under some very harsh (denaturing) conditions (e.g., 8 M urea, 80% acetonitrile) and at temperatures as low as 4 degrees C and up to 80 degrees C but severely hampered by guanidine hydrochloride and methanol. These rather unique features make Lys-N a good candidate for a variety of applications, such as membrane proteomics and possibly H/D exchange mass spectrometry. Additionally, we show that Lys-N is capable of, in contrast to trypsin or Lys-C, cleaving adjacent to mono- and dimethylated lysines, making it a good candidate for targeted epigenetic analysis of for instance histones. |
| spellingShingle | Taouatas, N Heck, A Mohammed, S Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions. |
| title | Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions. |
| title_full | Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions. |
| title_fullStr | Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions. |
| title_full_unstemmed | Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions. |
| title_short | Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions. |
| title_sort | evaluation of metalloendopeptidase lys n protease performance under different sample handling conditions |
| work_keys_str_mv | AT taouatasn evaluationofmetalloendopeptidaselysnproteaseperformanceunderdifferentsamplehandlingconditions AT hecka evaluationofmetalloendopeptidaselysnproteaseperformanceunderdifferentsamplehandlingconditions AT mohammeds evaluationofmetalloendopeptidaselysnproteaseperformanceunderdifferentsamplehandlingconditions |