Protein Sequencing: MUDPIT

Mass Spectrometry and Complex Protein Mixture Sequencing (1-3000 proteins)

MUDPIT (multidimensional protein identification technology) is the method of choice for complex protein sample analysis in which more elaborate separation techniques are needed. For MUDPIT analysis strong cation exchange resin (SCX), which binds positively charged compounds, is packed in tandem to the RP-C18 resin. SCX resin first binds all peptides before they encounter the C18 material. The peptides are then eluted off the SCX using ammonium salts in a stepwise manner, starting at low salt concentration step and ending with a high salt concentration step. After each salt step some peptides will be released from the SCX resin and will bind the RP-C18 material. This salt step is then followed by a cycle of organic gradient to elute off the peptides from the C18 resin and into the mass spectrometer for sequencing. For more complex samples more salt steps can be added to the method to increase the separation capability of the setup.

Recent method developments in peptide separation are using alternative separation strategies to SCX to improve peak separation and hence increase peptide identifications for Mudpit. One encouraging method is the use of "high-pH-reverse-phase" separation. Similar to low pH reverse phase separation techniques currently used in LC-MS, the high pH equally generates high peak resolution for peptides. We have observed that the use of this method in replacement of SCX in Mudpit analysis increases peptide identifications in similar Mudpit runs by the factor of two.