At the UVic Genome BC Proteomics Centre, protein identification is performed using bottom-up mass spectrometric (MS) approaches in which peptides are analyzed by accurately determining mw and amino acid sequence by tandem mass spectrometry. Our primary workflows involve proteolytic digestion of proteins (in-gel or in solution) followed by either direct MS analysis or MS analysis after 1D or 2D liquid chromatography separation of peptides. For MS analysis of samples, both electrospray (ESI) and MALDI ionization modes are routinely used.
We have developed numerous methods for comprehensive and highly sensitive identification of proteins including the combination of peptide mass fingerprinting and tandem MS (7). For example improved peptide coverage and increased confidence can be achieved by combining high field FT-MS with post acquisition data analysis, which includes per-scan recabilibration of data followed by mixture mass fingerprinting with sub-ppm mass accuracy (2). Increased sensitivity of protein ID can be obtained by skipping gel staining steps and using MALDI to direct tandem MS data acquisition by ESI.
- whole lane analysis of 1D SDS-PAGE (GeLC/MS) (5)
- increased sensitivity without staining (3)
- automated in gel digestion (Progest, Genomic Solutions) (3)
- strong cation exchange fractionation
- free flow electrophoresis (FFE, Becton Dickinson)
- tandem MS/MS analysis (1-7)
- nanoflow (<300 nl/min) reversed phase HPLC
- peptide mass fingerprinting (PMF) (7)
- Proc J.L., Kuzyk M.A., Hardie D.B., Yang J., Smith D.S., Jackson A.M., Parker C.E., Borchers C.H. A Quantitative Study of the Effects of Chaotropic Agents, Surfactants, and Solvents on the Digestion Efficiency of Human Plasma Proteins by Trypsin. J.Prot. Res. (2010).
- Danell, R., Ouvry-Patat, S.A., Scarlett, C.O., Speir, J.P., Borchers C.H., DAta SElf-Recalibration and Mixture Mass Fingerprint Searching (DASER-MMF) to Enhance Proteomics Identifications from Complex Mixtures. JASMS. (2008) 19:1914-1925.
- Parker, C. E.; Warren, M. R.; Loiselle, D., Dechiva, N.N., Scarlett, C.O., Borchers, C.H., Identification of Components of Protein Complexes. In: Methods in Molecular Biology, Patterson, W. C. (ed.) Humana Press, NJ, Vol. 103, 117-151 (2005)
- Loiselle, D.R., Thelin, W.R., Parker, C.E., Dicheva, N.N., Kesner, B.A., Mocanu, V., Wang, F., Milgram, S.L., Warren, M.R.E., Borchers, C.H., Improved Protein Identification through the Use of Unstained Gels. Journal of Proteome Research. (2005) 4(3): 992-997.
- Hall, M.C., Torres, M.P., Schroeder, G., Borchers, C.H., Mnd2 and Swm1 are Core Subunits of the Saccharomyces cerevisiae Anaphase-Promoting Complex. J. Biol. Chem.(2003) 19: 16698-16705.
- Kast, J., Parker, C.E., Drift, K.v.d., Dial, J.M., Milgram, S.L., Wilm, M., Howell, M., Borchers, C.H., Matrix-assisted laser desorption/ionization directed nano-electrospray ionization tandem mass spectrometric analysis for protein Identification. Rap. Commun. Mass Spectrom. (2003) 17: 1825-1834.
- Borchers, C., Peter, J.F., Hall, M.C., Kunkel, T.A., Tomer, K.B., Identification of In-Gel Digested Proteins by Complementary Peptide-Mass Fingerprinting and Tandem Mass Spectrometry Data obtained on an Electrospray Ionization Quadrupole Time-of-flight Mass Spectrometer. Analytical Chemistry. (2000) 72(6): 1163-1168.