Localization of Disulfide Bonds in Ribonuclease Using Low pH Trypsin and LC-ESI-QTOF-MS
We evaluated a method to localize disulfide bonds in bovine pancreatic ribonuclease (RNase) by applying low pH trypsin protein digestion with a bottom-up LC-ESI-QTOF-MS approach. The goal was to minimize disulfide bond scrambling during sample preparation. By using N-ethylmaleimide (NEM) for alkylat...
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| Language: | deu |
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Swiss Chemical Society
2024-12-01
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| Series: | CHIMIA |
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| Online Access: | https://www.chimia.ch/chimia/article/view/7475 |
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| author | Amélie Bornex Saša M. Miladinović |
| author_facet | Amélie Bornex Saša M. Miladinović |
| author_sort | Amélie Bornex |
| collection | DOAJ |
| description | We evaluated a method to localize disulfide bonds in bovine pancreatic ribonuclease (RNase) by applying low pH trypsin protein digestion with a bottom-up LC-ESI-QTOF-MS approach. The goal was to minimize disulfide bond scrambling during sample preparation. By using N-ethylmaleimide (NEM) for alkylation of free cysteines, we achieved 92% sequence coverage and successfully identified the native disulfide bonds between specific cysteine residues. However, scrambled disulfide bonds were also observed. Our results indicate that while this low pH digestion method helps preserve native disulfide bonds, further refinement is needed to fully prevent disulfide bond rearrangement during sample preparation.
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| format | Article |
| id | doaj-art-e072ba65d2df4a938d18aa4dcd391929 |
| institution | OA Journals |
| issn | 0009-4293 2673-2424 |
| language | deu |
| publishDate | 2024-12-01 |
| publisher | Swiss Chemical Society |
| record_format | Article |
| series | CHIMIA |
| spelling | doaj-art-e072ba65d2df4a938d18aa4dcd3919292025-08-20T02:29:51ZdeuSwiss Chemical SocietyCHIMIA0009-42932673-24242024-12-01781210.2533/chimia.2024.881Localization of Disulfide Bonds in Ribonuclease Using Low pH Trypsin and LC-ESI-QTOF-MSAmélie Bornex0Saša M. Miladinović1HES-SO Valais-Wallis, University of Applied Sciences, Institute of Life Sciences, CH-1950 Sion; Lonza AG, CH-3930 VispHES-SO Valais-Wallis, University of Applied Sciences, Institute of Life Sciences, CH-1950 SionWe evaluated a method to localize disulfide bonds in bovine pancreatic ribonuclease (RNase) by applying low pH trypsin protein digestion with a bottom-up LC-ESI-QTOF-MS approach. The goal was to minimize disulfide bond scrambling during sample preparation. By using N-ethylmaleimide (NEM) for alkylation of free cysteines, we achieved 92% sequence coverage and successfully identified the native disulfide bonds between specific cysteine residues. However, scrambled disulfide bonds were also observed. Our results indicate that while this low pH digestion method helps preserve native disulfide bonds, further refinement is needed to fully prevent disulfide bond rearrangement during sample preparation. https://www.chimia.ch/chimia/article/view/7475Bottom-up proteomicsDisulphide bondsLC-MSLow pH trypsin |
| spellingShingle | Amélie Bornex Saša M. Miladinović Localization of Disulfide Bonds in Ribonuclease Using Low pH Trypsin and LC-ESI-QTOF-MS CHIMIA Bottom-up proteomics Disulphide bonds LC-MS Low pH trypsin |
| title | Localization of Disulfide Bonds in Ribonuclease Using Low pH Trypsin and LC-ESI-QTOF-MS |
| title_full | Localization of Disulfide Bonds in Ribonuclease Using Low pH Trypsin and LC-ESI-QTOF-MS |
| title_fullStr | Localization of Disulfide Bonds in Ribonuclease Using Low pH Trypsin and LC-ESI-QTOF-MS |
| title_full_unstemmed | Localization of Disulfide Bonds in Ribonuclease Using Low pH Trypsin and LC-ESI-QTOF-MS |
| title_short | Localization of Disulfide Bonds in Ribonuclease Using Low pH Trypsin and LC-ESI-QTOF-MS |
| title_sort | localization of disulfide bonds in ribonuclease using low ph trypsin and lc esi qtof ms |
| topic | Bottom-up proteomics Disulphide bonds LC-MS Low pH trypsin |
| url | https://www.chimia.ch/chimia/article/view/7475 |
| work_keys_str_mv | AT ameliebornex localizationofdisulfidebondsinribonucleaseusinglowphtrypsinandlcesiqtofms AT sasammiladinovic localizationofdisulfidebondsinribonucleaseusinglowphtrypsinandlcesiqtofms |