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Multidimensional Protein Identification Technology (MudPIT)

We utilize a method named Multidimensional Protein Identification Technology (MudPIT) to analyze the proteomes of organisms. MudPIT is a chromatography-based proteomic technique where a complex peptide mixture is prepared from a protein sample and loaded directly onto a triphasic microcapillary column packed with reversed phase, strong cation exchange, and reversed phase HPLC grade materials. Once the complex peptide mixture is loaded onto the triphasic microcapillary column, this column is placed directly in-line with a tandem mass spectrometer. The tandem mass spectrometry data generated from a MudPIT run is then searched to determine the protein content of the original sample.

mudpit steps Sample Prep Digestion Multidimensional Chromatography Tandem Mass Spectrometry Database Matching Data Analysis Default

MudPIT is a robust and widely accepted method for protein identification from a wide variety of samples. MudPIT has been proven to be an excellent tool for both qualitative and quantitative proteomic analyses. Through on-line two dimensional HPLC, complex peptides mixtures can be well separated. Recent data shows that even for a relatively simple sample, better results are obtained from MudPIT, compared to single dimension LC method.

Washburn M.P. (2004) Utilisation of proteomics datasets generated via multidimensional protein identification technology (MudPIT). Brief Funct. Genomic Proteomic, 3(3):280-6. Abstract

Swanson S.K., Washburn M.P. (2005) The continuing evolution of shotgun proteomics. Drug Discov. Today, 10(10):719-25. Abstract

Florens, L., & Washburn, M.P. (2006) Proteomic analysis by Multidimensional Protein Identification Technology. Methods Mol. Biol., 328, 159-175. Abstract

Fournier, M.L., Gilmore, J.M., Martin-Brown S.A., & Washburn, M.P. (2007) Multidimensional separations-based shotgun proteomics. Chem. Rev., 107(8):3654-86. Abstract

Washburn, M.P. (2008) Sample preparation and in-solution protease digestion of proteins for chromatography-based proteomic analysis. Curr. Protoc. Protein Sci., Chapter 23:Unit 23.6.1-23.6.11. Abstract

Florens, L., Korfali, N. & Schirmer, E.C. (2008) Subcellular fractionation and proteomics of nuclear envelopes. Methods Mol. Biol., 432, 117-137. Abstract

Korfali, N., Fairley, E.A., Swanson, S.K., Florens, L., & Schirmer, E.C. (2009) Use of sequential chemical extractions to purify nuclear membrane proteins for proteomics identification. Methods Mol. Biol., 528, 201-225. Abstract

Swanson, S.K., Florens, L., and Washburn, M.P. (2009) Generation and analysis of multidimensional protein identification technology datasets. Methods Mol. Biol., 492:1-20. Abstract