Abstract
Nanoflow liquid chromatography coupled with nano-electrospray (nanoLC-MS) is the method of choice for sensitive peptide and protein analysis for proteomics research. We recently reported on a new microfluidic platform (cHiPLC-nanoflex) for nanoLC-MS applications. In addition to delivering easy-to-use, dead-volume-free connections to microfluidic devices, the platform's cHiPLC columns provide excellent column-to-column separation reproducibility. We have reported previously(1) that while increasing only gradient time or column length can improve peak capacity, using a longer column in combination with increasing gradient time is the most effective way of obtaining higher peak capacity. In this presentation we report on how the dead volume free connections of the cHiPLC Nanoflex platform allow for the coupling of two 15 cm long chip columns for improved resolution. The proposed set-up makes it easy to switch between single and dual column mode depending on application requirements. An additional advantage of using two separate 15 cm columns rather than one 30cm column is that sample matrix components that are not retained on the first column (e.g. salts) can be directed to waste, instead of entering the nanospray source/mass spectrometer. We have investigated the effect of increased resolution through doubling column length on the number of peptides/proteins identified in a digested cytosolic fraction of a human cell lysate using various gradient lengths. An ABSCIEX TripleTOF 5600™ MS was used with an Information Dependent Acquisition method consisting of a TOF MS survey scan at 30 000 resolution, followed by 20 MS/MS in a second at >15 000 resolution. All data was processed using ProteinPilot™ Software 4.0 with integrated false discovery rate (FDR) analysis. 1 Remco van Soest*, David W. Neyer; Jia Eng Siow, and Phil Paul, Eksigent Technologies, Dublin, CA; poster ASMS 2008: “Improving resolution in nanoLC separations for proteomics using ultra high pressures”.