MudPIT combines both a cation exchange pre-fractionation and RP HPLC separation (1) of tryptic peptides to analyze an entire proteome of a cell or tissue type protein extract. The approach in use in the Keck MS & Proteomics Resource is to use a dual enzymatic digestion (Lys-C followed by trypsin (2)) in order to increase the number of peptides observed. The peptides are separated using strong cation exchange on an AB Vision Workstation, followed by LC-MS/MS and protein identification on each fraction using the AB QSTAR Elite or the Thermo Scientific LTQ-Orbitrap XL mass spectrometer.
- Sample Amount=50 to 200μg
- Wolters, D.A., Washburn, M.P., and Yates, J.R. (2001) An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics. Anal. Chem., 73(23):5683 - 5690.
- Choudhary, G., Wu, S., Shieh, P., and Hancock, W.S. (2003) Multiple Enzymatic Digestion for Enhanced Sequence Coverage of Proteins in Complex Proteomic Mixtures Using Capillary LC with Ion Trap MS/MS. J. Proteome Res., 2:59-67.
- Stone, K.L. and Williams, K.R (2009) Reverse-Phase HPLC Separation of Enzymatic Digests of Proteins in The Protein Protocols Handbook, 3rd edition (Walker, J., ed.) 937-946.