Isolation of acetylated and unmodified protein N-Terminal peptides by strong cation exchange chromatographic separation of TrypN-digested peptides
| Autor: | Juri Rappsilber, Hsin Yi Chang, Chih-Hsiang Chang, Yasushi Ishihama |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Proteomics
Lysis LC/MS/MS Ion chromatography SLS sodium N-lauroylsarcosinate N terminomics Biochemistry COFRADIC combined fractional diagonal chromatography Analytical Chemistry chemistry.chemical_compound Tandem Mass Spectrometry Trypsin Chromatography High Pressure Liquid 0303 health sciences CAA 2-chloroacetamide Chemistry Escherichia coli Proteins 030302 biochemistry & molecular biology Technological Innovation and Resources acetylated N-terminal peptide Acetylation Terminal amine isotopic labeling of substrates Chromatography Ion Exchange SCX strong cation exchange chromatography shotgun proteomics Proteome TCEP SDC sodium deoxycholate N-terminal peptide enrichment Protein digestion HEK293T TCEP tris(2-carboxyethyl)phosphine StageTip stop and go extraction tip protein N terminus TFA trifluoroacetic acid 03 medical and health sciences TrypN HYTANE hydrophobic tagging-assisted N-termini enrichment Trifluoroacetic acid Humans Shotgun proteomics Molecular Biology 030304 developmental biology Chromatography PTS phase-transfer surfactants ACN acetonitrile HEK293 Cells MS mass spectrometry ChaFRADIC charge-based fractional diagonal chromatography TAILS terminal amine isotopic labeling of substrates SCX Peptides Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride |
| Zdroj: | Chang, C-H, Chang, H-Y, Rappsilber, J & Ishihama, Y 2021, ' Isolation of acetylated and unmodified protein N-Terminal peptides by strong cation exchange chromatographic separation of TrypN-digested peptides ', Molecular and Cellular Proteomics, vol. 20, 100003 . https://doi.org/10.1074/mcp.TIR120.002148 Molecular & Cellular Proteomics : MCP |
| DOI: | 10.1074/mcp.TIR120.002148 |
| Popis: | 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. Graphical Abstract Highlights • Isolation of protein N-terminal peptides without chemical reaction • Both N-acetylated and unmodified protein N termini can be isolated • Two-step isolation consisting of TrypN digestion and strong cation exchange separation • Less than 3% contamination with internal peptides In Brief N-acetylated and unmodified protein N-terminal peptides were successfully isolated by strong cation exchange chromatographic separation of TrypN-digested peptides without chemical reaction. From 20 μg of human HEK293T cell lysate proteins, 1550 acetylated and 200 unmodified protein N-terminal peptides were 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|