Integrated microfluidic devices with enhanced separation performance: application to phosphoproteome analyses of...

This work reports on the application of a microfluidic device integrating nanoscale LC to nanoelectrospray MS (nano-LC-chip-MS) for the analysis of complex protein digests. Peak profile analyses of more than 700 peptide ions, reproducibly detected across replicate nano-LC-chip-MS runs (n = 5), indicated that the system provided RSD values of 0.24% on retention time, +/- 30 ppm on m/z measurement and +/- 30% variation on intensity over three orders of magnitude. RP adsorbant media with different alkyl chains and particle size packed in both trapping and separation channels were investigated to improve the chromatographic performance of this system. A two-fold improvement in chromatographic peak capacity was achieved using microfluidic devices comprising a 5 mircrom C3 trap with 2.5 microm C18 trap separation channel compared to the traditional 5 microm C18 stationary phase. Enhanced sample selectivity for the identification of phosphopeptides was obtained by combining immobilized metal affinity media prior to peptide separation on the RP microfluidic device. This system was evaluated in the context of differential phosphoproteome analyses to identify changes in signaling events and protein expression of human monocytes following the administration of phorbol ester.