Pressurized pepsin digestion in proteomics: an automatable alternative to trypsin for integrated top-down bottom-up proteomics.

Molecular & Cellular Proteomics : MCP
Daniel Lopez-FerrerLjiljana Pasa-Tolić

Abstract

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the resu...Continue Reading

References

May 5, 1982·Journal of Molecular Biology·J Kyte, R F Doolittle
Jan 17, 1995·Proceedings of the National Academy of Sciences of the United States of America·B K ShoichetB W Matthews
Apr 25, 2001·Rapid Communications in Mass Spectrometry : RCM·S SunS Liu
May 2, 2002·Proceedings of the National Academy of Sciences of the United States of America·Sang-Won LeeRichard D Smith
Mar 28, 2003·Analytical Chemistry·Jan HavlisAndrej Shevchenko
Apr 30, 2003·Rapid Communications in Mass Spectrometry : RCM·Gordon W Slysz, David C Schriemer
Dec 31, 2003·Analytical Chemistry·Yufeng ShenRichard D Smith
Mar 15, 2005·Analytical Chemistry·Gordon W Slysz, David C Schriemer
Oct 11, 2005·Journal of Proteome Research·D López-FerrerJ Vázquez
Jul 18, 2006·Analytical Chemistry·Wenqing ShuiXian Chen
Aug 8, 2006·Journal of Proteome Research·Gordon W SlyszDavid C Schriemer
Nov 1, 2006·Journal of the American Society for Mass Spectrometry·Maolian ChenRonald Wetzel
Feb 3, 2007·Journal of Proteome Research·Seema SharmaLjiljana Pasa-Tolić
Dec 11, 2007·Journal of Proteome Research·Jesse RodriguezPavel A Pevzner
May 2, 2008·Molecular & Cellular Proteomics : MCP·Leonie F WaandersMatthias Mann
Jul 9, 2008·Journal of Proteome Research·Daniel López-FerrerRichard D Smith
Aug 9, 2008·Journal of Proteome Research·Daniel López-FerrerRichard D Smith
Dec 20, 2008·Journal of the American Society for Mass Spectrometry·Hui-Min ZhangAlan G Marshall
Jan 1, 2009·Molecular & Cellular Proteomics : MCP·Benjamin RietschelAnsgar Poetsch
May 12, 2009·Analytical Chemistry·Si WuLjiljana Pasa-Tolić
Jun 25, 2009·Analytical Chemistry·Daniel López-FerrerRichard D Smith
Oct 23, 2009·Proceedings of the National Academy of Sciences of the United States of America·Leonie F WaandersMatthias Mann
Dec 3, 2009·Analytica Chimica Acta·Andrew J Percy, David C Schriemer
Feb 2, 2010·Journal of Proteome Research·Danielle L SwaneyJoshua J Coon
Mar 3, 2010·Rapid Communications in Mass Spectrometry : RCM·Hyo-Jik YangJeongkwon Kim
May 18, 2010·International Journal of Mass Spectrometry·Aleksey V TolmachevRichard D Smith
Nov 1, 1994·Journal of the American Society for Mass Spectrometry·J K EngJ R Yates

❮ Previous
Next ❯

Citations

Aug 24, 2011·Molecular & Cellular Proteomics : MCP·Ruijun TianDaniel Figeys
Jul 12, 2014·Annual Review of Analytical Chemistry·Zhaorui ZhangLjiljana Paša-Tolić
Jan 10, 2012·Current Opinion in Chemical Biology·A F Maarten Altelaar, Albert J R Heck
Aug 1, 2012·Mass Spectrometry Reviews·Eva Rodríguez-Suárez, Anthony D Whetton
Oct 16, 2012·Biochimica Et Biophysica Acta·Joomi AhnJohn R Engen
Apr 29, 2016·Nature Protocols·Piero GiansantiAlbert J R Heck
Oct 20, 2018·Lab on a Chip·Camila D M CamposSteven A Soper
Apr 14, 2012·Chemical Society Reviews·Thomas E AngelRichard D Smith
Jul 25, 2017·Expert Review of Proteomics·Upendra K KarJulian P Whitelegge
Oct 7, 2020·Molecular & Cellular Proteomics : MCP·Diana SamodovaJesper V Olsen
Jan 17, 2021·Molecular & Cellular Proteomics : MCP·Diana SamodovaJesper V Olsen
Aug 3, 2017·Journal of Chromatography. a·Lian YiWei-Jun Qian
Feb 2, 2013·Journal of Proteome Research·Linda SwitzarWilfried M A Niessen
Nov 4, 2011·Analytical Chemistry·Hu ZhouDaniel Figeys

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.