MudPIT (multidimensional protein identification technology) is a non-gel approach for the identification of proteins from complex mixtures. The technique consists of a 2-dimensional chromatography separation, prior to electrospray mass spectrometry. By exploiting a peptide's unique physical properties of charge and hydrophobicity, complex mixtures can be separated prior to sequencing by tandem MS. The first dimension is normally a strong cation exchange (SCX) column, as these have high loading capacities. The second dimension is reverse phase chromatography (RP), which complements the SCX as it is efficient at removing salts and has the added advantage of being compatible with electrospray mass spectrometry.

Sample preparation is relatively straightforward, the samples are denatured, the cysteines reduced and alkylated and the proteins digested with a protease such as trypsin. The samples are then acidified and loaded onto the SCX column (see diagram below). Charged peptides bind to the SCX column, whereas any uncharged peptides pass through and bind to a reverse phase trap column. The peptides are then eluted from the trap column onto an analytical RP column, using a reverse phase gradient, separated and eluted into a tandem mass spectrometer. Peptide fragmentation data is then obtained to identify the peptides and hence the proteins from which they are derived. In the next step, salt at a particular concentration is injected onto the SCX column, displacing further peptides from it onto the RP trap column. Salt is removed by washing and again an analytical RP separation is performed and the eluting peptides analysed by mass spectrometry. Incremental increases of salt are used (salt step gradient from around 0 - 200 mM). The end result is multiple protein identifications from each salt step.