Preparation of 20-μm-i.d. Silica-Based Monolithic Columns and Their Performance for Proteomics Analyses

We describe the preparation and performance of high-efficiency 70 cm × 20 μm i.d. silica-based monolithic
capillary LC columns. The monolithic columns at a
mobile-phase pressure of 5000 psi provide flow rates of
∼40 nL/min at a linear velocity of ∼0.24 cm/s. The
columns provide a separation peak capacity of ∼420 in
conjunction with both on-line coupling with microsolid-phase extraction and nanoelectrospray ionization-mass
spectrometry. Performance was evaluated using a Shewanella oneidensis tryptic digest, and ∼15-amol detection limits for peptides were obtained using a conventional
ion trap and MS/MS for peptide identification. The
sensitivity and separation efficiency enabled the identification of 2367 different peptides covering 855 distinct S.
oneidensis proteins from a 2.5-μg tryptic digest sample
in a single 10-h analysis. The number of identified
peptides and proteins approximately doubled when the
effective separation time was extended from 200 to 600
min. The number of identified peptides increased from
32 to 390 as the injection amount was increased from
0.5 to 100 ng. Both the run-to-run and column-to-column
reproducibility for proteomic analyses were also evaluated.