Proteomic investigations using a double-vented tetraphasic continuous column approach to MudPIT analysis

ANYL 23

Andrew Guzzetta, guzzetta@stanford.edu and Allis Chien. Stanford University Mass Spectrometry, Stanford University, 380 Roth Way, Keck 328, Stanford, CA 94305-5080
A double-vented serial tetraphasic capillary column approach is applied to proteomic MudPIT-type analysis using extended length capillary reverse-phase columns. The heart of the tetraphasic device consists of a triphasic MudPIT trap located upstream of a venting tee. The trap is followed by a 60 cm high-resolution capillary column. A conventional high-flow HPLC is used to develop gradients at standard flow rates and pressures. The double-vented triphasic MudPT trapping device relieves the capillary separation column from the salt burden during the on-line cation-exchange portion of the analysis. Since MudPIT has the potential of splitting peptides between salt steps we have also looked into the potential of differential combination of salt steps. Toward this end we have explored the proteomic advantage of combining data from repeat identical MudPIT runs and have compared this to an experiment in which we have combined repeat MudPIT runs that differed in their salt step elutions.