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

Research output: Contribution to journalArticle

31 Citations (Scopus)

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 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
Volume10
Issue number2
DOIs
Publication statusPublished - 2011 Feb

Fingerprint

Pepsin A
Proteomics
Trypsin
Digestion
Online Systems
Proteome
Monkeypox virus
Pressure
Peptides
Proteins
Workflow
Biological systems
Post Translational Protein Processing
Viruses
Protein Isoforms
Throughput

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Cite this

Pressurized pepsin digestion in proteomics : An automatable alternative to trypsin for integrated top-down bottom-up proteomics. / López-Ferrer, Daniel; Petritis, Konstantinos; Robinson, Errol W.; Hixson, Kim K.; Tian, Zhixin; Lee, Jung Hwa; Lee, Sang-Won; Tolić, Nikola; Weitz, Karl K.; Belov, Mikhail E.; Smith, Richard D.; Paša-Tolić, Ljiljana.

In: Molecular and Cellular Proteomics, Vol. 10, No. 2, 02.2011.

Research output: Contribution to journalArticle

López-Ferrer, D, Petritis, K, Robinson, EW, Hixson, KK, Tian, Z, Lee, JH, Lee, S-W, Tolić, N, Weitz, KK, Belov, ME, Smith, RD & 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, vol. 10, no. 2. https://doi.org/10.1074/mcp.M110.001479
López-Ferrer, Daniel ; Petritis, Konstantinos ; Robinson, Errol W. ; Hixson, Kim K. ; Tian, Zhixin ; Lee, Jung Hwa ; Lee, Sang-Won ; Tolić, Nikola ; Weitz, Karl K. ; Belov, Mikhail E. ; Smith, Richard D. ; Paša-Tolić, Ljiljana. / Pressurized pepsin digestion in proteomics : An automatable alternative to trypsin for integrated top-down bottom-up proteomics. In: Molecular and Cellular Proteomics. 2011 ; Vol. 10, No. 2.
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