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

Daniel López-Ferrer, Konstantinos Petritis, Errol W. Robinson, Kim K. Hixson, Zhixin Tian, Jung Hwa Lee, Sang-Won Lee, Nikola Tolić, Karl K. Weitz, Mikhail E. Belov, Richard D. Smith, Ljiljana Paša-Tolić

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31 Citations (Scopus)


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 results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a Re-Play configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.

Original languageEnglish
JournalMolecular and Cellular Proteomics
Issue number2
Publication statusPublished - 2011 Feb


ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Cite this

López-Ferrer, D., Petritis, K., Robinson, E. W., Hixson, K. K., Tian, Z., Lee, J. H., Lee, S-W., Tolić, N., Weitz, K. K., Belov, M. E., Smith, R. D., & Paša-Tolić, L. (2011). Pressurized pepsin digestion in proteomics: An automatable alternative to trypsin for integrated top-down bottom-up proteomics. Molecular and Cellular Proteomics, 10(2).