title: Uncovering immobilized trypsin digestion features from large-scale proteome data generated by high-resolution mass spectrometry.
abstract: Immobilized trypsin produces very fast protein digestion, which is attractive for application to high throughput bottom-up proteomics. While there is a rich literature on the preparation of immobilized trypsin, there are very few studies that investigate its application to complex proteomic samples. In this work, we compared solution-phase trypsin with trypsin immobilized on magnetic microspheres for digestion of two complex proteomes, Escherichia coli and the MCF7 cell line. The digests were separated by HPLC, and detected with a Q-Exactive mass spectrometer, which generated high resolution and high quality parent- and fragment-ion mass spectra. The data were analyzed using MaxQuant. We make several conclusions about the features of immobilized trypsin digestion of complex proteomes. First, both immobilized and solution-phase trypsin generate peptides that sample the same protein pool. Second, immobilized trypsin can digest complex proteomes two orders of magnitude faster than solution-phase trypsin while retaining similar numbers of protein identifications and proteome depth. Digestion using immobilized trypsin for 5-min produces a similar number of missed cleavages as solution-based trypsin digestion for 4-h; digestion using immobilized trypsin for 20-min produces a similar number of missed cleavages as solution-based trypsin digestion for 12-h. Third, immobilized trypsin produces quantitatively reproducible digestion of complex proteomes. Finally, there is small but measurable loss of peptide due to non-specific adsorption to the immobilization matrix. This adsorption generates a bias against detection of basic peptides.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Guijie
authors.initials: G
authors.lastname: Zhu
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Yan
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Si
authors.initials: S
authors.lastname: Mou
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. Electronic address: ndovichi@nd.edu.
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D000327:Adsorption; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D020543:Proteome; D014357:Trypsin
keywords: Bottom-up proteomics; Complex proteome; High resolution mass spectrometry; Immobilized trypsin; Reproducibility