Multi-Dimensional Protein Identification Technology (MudPIT)
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.
Both MS systems are equipped with Waters nanoAcquity UPLC systems (3) which enable 75 µm x 25 cm reverse phase columns to be used, thereby increasing the peptide separation due to the longer column length (25 vs 15 cm). The data is analyzed using the Mascot MudPIT search in Mascot Daemon. The results are loaded into YPED for online viewing. The MudPIT approach is also used for SILAC labeled samples.
- 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.