Low-Attomole Electrospray Ionization MS and MS/MS Analysis of Protein Tryptic Digests Using 20-μm-i.d. Polystyrene-Divinylbenzene Monolithic Capillary Columns.

This work explores the use of 20-μm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase composition were optimized for chromatographic performance leading to efficiencies over 100000 plates/m for peptide separations. High mass sensitivity (∼10 amol of peptides) in the MS and MS/MS modes using an ion trap MS was found, a factor of up to 20-fold improvement over 75-μmi.d. nanocolumns. A wide linear dynamic range (∼4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradient nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of ∼1000 cells injected, is presented. [ABSTRACT FROM AUTHOR]