High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies

The use of tetrahydrofuran/decanol as porogens for the fabrication of micro pellicular poly( styrene/divinylbenzene) monoliths enabled the rapid and hi ghly efficient separation of peptides and proteins by reversed-phase high-p erformance liquid chromatography RP-HPLC). In contrast to conventional, gra nular, porous stationary phases, in which the loading capacity is a functio n of molecular mass, the loadability of the monoliths both for small peptid es and large proteins was within the 0.4-0.9-pmol range for a 60- x 0.2-mm capillary column, Lower limits of detection obtained by measuring UV-absorb ance at 214 mn with a 3-nl capillary detection cell were 500 amol for an oc tapeptide and 200 amol for ribonuclease A. Upon reduction of the concentrat ion of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ioni zation mass spectrometry (ESI-RIS) at the cost of only a small decrease in separation efficiency. Detection Limits for proteins with ESI-RIS were in t he lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides an d proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferri n and by the analysis of membrane proteins related to the photosystem II an tenna complex of higher plants.