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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Sun, Liangliang Yan, Xiaojing Mou, Si Zhu, Guijie Dovichi, Norman J. |
| Description | Country affiliation: United States Author Affiliation: Sun L ( Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.); Zhu G ( Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.); Yan X ( Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.); Mou S ( Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.); Dovichi NJ ( Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA. Electronic address: ndovichi@nd.edu.) |
| 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. |
| ISSN | 00219673 |
| e-ISSN | 18733778 |
| DOI | 10.1016/j.chroma.2014.02.014 |
| Journal | Journal of Chromatography A |
| Volume Number | 1337 |
| Language | English |
| Publisher | Elsevier |
| Publisher Date | 2014-04-11 |
| Publisher Place | Netherlands |
| Access Restriction | Open |
| Subject Keyword | Proteome Trypsin Chemistry Adsorption Cell Line, Tumor Chromatography, High Pressure Liquid Enzymes, Immobilized Escherichia Coli Proteins Metabolism Magnetics Microspheres Proteolysis Reproducibility Of Results Spectrometry, Mass, Electrospray Ionization Tandem Mass Spectrometry Research Support, N.i.h., Extramural Discipline Analytical Chemistry |
| Content Type | Text |
| Resource Type | Article |
| Subject | Organic Chemistry Medicine Analytical Chemistry Biochemistry |
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