Integrated MICROFASP method with CZE based fractionation technique and nanoRPLC-ESI-MS/MS for comprehensive proteomics analysis of submicrogram sample

We report a loss-less two dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for comprehensive proteomics analysis of submicrogram sample. Protein digest was injected into the linear polyacrylamide coated capillary followed by CZE separation. The schemes to collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials followed by nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimized sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5,231 protein groups from 500 ng of a HeLa protein digest within 11.5 hours (90 min CZE fractionation plus 10 hours LC-MS analysis). Finally, the developed platform was used to analysis the protein digest prepared by MICROFASP method with 1 µg cell lysate as starting material. 3796 (N=2, RSD=4.95%) protein groups and 20,577(N=2, RSD=7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 hours. The results indicated that combination of MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of submicrogram biological sample.