PNNL

Abstract

We report on the preparation and performance of a high efficiency 70 cm ´ 20 µm i.d. silica-based monolithic capillary column. With a mobile phase delivery pressure of 5000 psi, this monolithic column provides flow rates as low as ~40 nL/min at an LC linear velocity of ~0.24 cm/s. The resultant columns provided a separation peak capacity of ~420 under conditions of on-line coupling micro solid phase extraction (SPE) and nanoelectrospray ionization (ESI) mass spectrometry (MS) for a Shewanella oneidensis tryptic digest. A sensitivity of ~15 attomole for detection of peptides was obtained when a conventional ion trap MS/MS was used for the detection. The sensitivity and separation efficiency of this column enabled identification of 2367 different peptides from 855 S. oneidensis distinct proteins from a 2.5 µg tryptic digest sample in a single 10-h analysis by nanoLC/MS/MS. The run-to-run and column-to-column reproducibility was investigated for proteomic analyses.