Comparing monolithic and microparticular capillary columns for the separation and analysis of peptide mixtures by...

A mixture of ten proteins was trypsinized and injected onto poly-(styrene-divinylben-zene) monolithic columns (60 x 0.20 or 0.10 mm ID) and a column packed with C18 silica particles (75 x 0.075 mm ID), respectively. The columns were eluted at 200-2000 nL/min with gradients of ACN in 0.050% TFA. Eluting peptides were detected by ESI-MS/MS and subsequently identified by database searching. The 100 microm ID monolithic column showed the highest cumulative Mowse scores based on the highest ion scores for the peptides and the largest number of identified peptides. It is shown that the number of identified peptides strongly depends on the dynamic range within the peptide mixture. In consequence, all proteins were identified in a mixture of relatively balanced analyte amounts (12.5-80 fmol) whereas only peptides for six out of ten proteins were found in a sample of high-dynamic range (0.65-270 fmol). The 100 microm monolithic column showed the highest reproducibility for peptide identifications in three consecutive runs. Depending on sample amount, 57-72% of the identified peptides were detectable in each of the three runs of triplicate analyses. The results demonstrate the high suitability of 100 microm monolithic columns for high-resolution peptide separations in proteomic research.