title: Pressurized pepsin digestion in proteomics: an automatable alternative to trypsin for integrated top-down bottom-up proteomics.
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 RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory, Richland, Washington 99354, USA. daniel.lopez-ferrer@pnl.gov
authors.forename: Daniel
authors.initials: D
authors.lastname: López-Ferrer
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Petritis
authors.forename: Errol W
authors.initials: EW
authors.lastname: Robinson
authors.forename: Kim K
authors.initials: KK
authors.lastname: Hixson
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Tian
authors.forename: Jung Hwa
authors.initials: JH
authors.lastname: Lee
authors.forename: Sang-Won
authors.initials: SW
authors.lastname: Lee
authors.forename: Nikola
authors.initials: N
authors.lastname: Tolić
authors.forename: Karl K
authors.initials: KK
authors.lastname: Weitz
authors.forename: Mikhail E
authors.initials: ME
authors.lastname: Belov
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Ljiljana
authors.initials: L
authors.lastname: Pasa-Tolić
mesh_terms: D001331:Automation; D001426:Bacterial Proteins / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods; D010434:Pepsin A / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D020592:Shewanella / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D011506:Proteins; D020543:Proteome; D014357:Trypsin; D010434:Pepsin A