PNNL

Abstract

In this study, high efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-um) C18 bonded particles provided a total peak capacity of ~1,000 for cellular proteolytic polypeptides when connecting to FTICR mass spectrometer through electrospray interface under composition gradient conditions at a pressure of 10,000 psi. Two-dimensional analyses from combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of >1 million polypeptide components, and simultaneously provided information for the identification of the seperated compents based upon the accurate mass tag concept previously described. For Deinococcus radiodurans tryptic digests, ~48.6000 polypeptides from ~60,7000 isotopic distributions have been detected in a single run. Large amount (e.g., 500 ug) of cellular proteolytic digests could be loaded on packed capillaries (e.g., 150-um inner diameter) without significant loss of speration efficiency. Pre-columns with suitable inner diameters were found useful for improving the elution reproducibility and without significant loss of seperation quality. Porous particle packled capillaries are more favorable than those containing non-porous particles because of their high sample capacity even for seperations of cellular proteolutic polypeptides with a moderate amount sample loading (e.g., 50 ug protein content).