title: Characterization of an immobilized enzyme reactor for on-line protein digestion.
abstract: Despite the developments for faster liquid chromatographic and mass spectral detection techniques, the standard in-solution protein digestion for proteomic analyses has remained relatively unchanged. The typical in-solution trypsin protein digestion is usually the slowest part of the workflow, albeit one of the most important. The development of a highly efficient immobilized enzyme reactor (IMER) with rapid performance for on-line protein digestion would greatly decrease the analysis time involved in a proteomic workflow. Presented here is the development of a silica based IMER for on-line protein digestion, which produced rapid digestions in the presence of organic mobile phase for both model proteins and a complex sample consisting of the insoluble portion of a yeast cell lysate. Protein sequence coverage and identifications evaluated between the IMER and in-solution digestions were comparable. Overall, for a yeast cell lysate with only a 10s volumetric residence time on-column, the IMER identified 507 proteins while the in-solution digestion identified 490. There were no significant differences observed based on identified protein's molecular weight or isoelectric point between the two digestion methods. Implementation of the IMER into the proteomic workflow provided similar protein identification results, automation for sample analysis, and reduced the analysis time by 15h.
authors.affiliation: Chemistry Department, University of North Carolina at Chapel Hill, NC 27599, United States.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Moore
authors.affiliation: Chemistry Department, University of North Carolina at Chapel Hill, NC 27599, United States.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Hess
authors.affiliation: Chemistry Department, University of North Carolina at Chapel Hill, NC 27599, United States. Electronic address: jj@unc.edu.
authors.forename: James
authors.initials: J
authors.lastname: Jorgenson
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized; D013058:Mass Spectrometry; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014357:Trypsin
chemical_list: D004800:Enzymes, Immobilized; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin
keywords: Digestion; IMER; Immobilized enzyme; Membrane proteins; Trypsin