High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics

We describe high-efficiency (peak capacities of similar to10(3)) nanoscale (using column inner diameters down to 15 mum) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74..5 pm were successfully packed with 3-mum C18-bonded porous (300-Angstrom pores) silica particles at a pressure of 18 000 psi. With a mobile-phase delivery pressure of 10 000 psi, these packed capillaries provided mobile-phase flow rates as low as similar to20 nL/min at LC linear velocities of similar to0.2 cm/s, which is near optimal for separation efficiency., To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 pm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 mum (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of similar to10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-mum-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C 18 particles with surface pores of 300-Angstrom versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC. seprations. Chromatographic, elution time, MS response intensity and Mass. measurement accuracy was examined between runs with a ingle column.(with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples. (various concentrations of cellular proteolytic peptides I) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.