دورية أكاديمية
Isolation of Acetylated and Unmodified Protein N-Terminal Peptides by Strong Cation Exchange Chromatographic Separation of TrypN-Digested Peptides.
| العنوان: | Isolation of Acetylated and Unmodified Protein N-Terminal Peptides by Strong Cation Exchange Chromatographic Separation of TrypN-Digested Peptides. |
|---|---|
| المؤلفون: | Chang CH; Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan., Chang HY; Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; Graduate Institute of Metabolism and Obesity Sciences, Taipei Medical University, Taipei, Taiwan., Rappsilber J; Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany; Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom., Ishihama Y; Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; Laboratory of Clinical and Analytical Chemistry, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan. Electronic address: yishiham@pharm.kyoto-u.ac.jp. |
| المصدر: | Molecular & cellular proteomics : MCP [Mol Cell Proteomics] 2021; Vol. 20, pp. 100003. Date of Electronic Publication: 2020 Nov 24. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Country of Publication: United States NLM ID: 101125647 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1535-9484 (Electronic) Linking ISSN: 15359476 NLM ISO Abbreviation: Mol Cell Proteomics Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: 2021- : [New York, NY] : Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Original Publication: Bethesda, MD : American Society for Biochemistry and Molecular Biology, [2002- |
| مواضيع طبية MeSH: | Peptides/*chemistry , Trypsin/*chemistry, Acetylation ; Chromatography, High Pressure Liquid ; Chromatography, Ion Exchange ; Escherichia coli Proteins/chemistry ; HEK293 Cells ; Humans ; Proteomics/methods ; Tandem Mass Spectrometry |
| مستخلص: | We developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N termini, and then, the N-terminal peptides with or without N-acetylation are separated from the internal peptides by strong cation exchange chromatography according to a retention model based on the charge/orientation of peptides. This approach was applied to 20 μg of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N termini registered in the Swiss-Prot database. Because this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N termini, including proteoforms with neo-N termini. Competing Interests: Conflict of interest Authors declare no competing interests. (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.) |
| معلومات مُعتمدة: | United Kingdom WT_ Wellcome Trust; 103139/Z/13/Z United Kingdom WT_ Wellcome Trust |
| فهرسة مساهمة: | Keywords: HEK293T; LC/MS/MS; N terminomics; N-terminal peptide enrichment; SCX; TrypN; acetylated N-terminal peptide; protein N terminus; shotgun proteomics |
| المشرفين على المادة: | 0 (Escherichia coli Proteins) 0 (Peptides) EC 3.4.21.4 (Trypsin) |
| تواريخ الأحداث: | Date Created: 20210131 Date Completed: 20220314 Latest Revision: 20240229 |
| رمز التحديث: | 20240229 |
| مُعرف محوري في PubMed: | PMC7857546 |
| DOI: | 10.1074/mcp.TIR120.002148 |
| PMID: | 33517145 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1535-9484 |
|---|---|
| DOI: | 10.1074/mcp.TIR120.002148 |