Parallel Channels-Multidimensional Protein Identification Technology.

Multidimensional protein identification (MudPIT), developed in the Yates Laboratory 20 years ago, is regarded as a powerful tool for proteomics research. Due to its remarkable online separation advantages, MudPIT has been widely used to facilitate discoveries in the field of proteomics research. However, it has one major disadvantage: the process of eluting peptides during strong cation exchange introduces salts, of different concentrations, into the mass spectrometer. Considering the sensitivity of the new generation of high-resolution mass spectrometers, developing a new version of MudPIT that could eliminate the introduction of salts in the elute would be a significant advancement to current technology. Herein, we developed a new, clean version of MudPIT called parallel channels-multidimensional protein identification technology (PC-MudPIT) to overcome this issue. In this design, the original biphasic trapping column was replaced by two parallel analytical column channels. We successfully averted the salt contamination yet retained all the other advantages of MudPIT. A total of 8161 and 7359 protein groups were identified from A549 whole cell lysate using PC-MudPIT and classic MudPIT, respectively. Moreover, we discovered the additional advantage that, in online mode, PC-MudPIT can also be used for an enrichment process of phosphopeptide identification. We identified a total 11453 phosphopeptides using PC-MudPIT and 7729 phosphopeptides using offline TiO2 enrichment followed by classic MudPIT. These advances indicate the possibility of other innovative applications of PC-MudPIT technology in deep proteome exploration.