Human proteome analysis by using reversed phase monolithic silica capillary columns with enhanced sensitivity.

We have developed one-dimensional liquid chromatography-tandem mass spectrometry systems with meter-scale reversed phase monolithic silica-C₁₈ capillary columns for human proteome analysis. When tryptic peptides from 4 μg HeLa cell lysate proteins were directly injected onto a 4-m, 100 μm i.d. monolithic silica-C₁₈ column and an 8-h gradient was applied at 500 nL/min, 41,319 non-redundant tryptic peptides from 5,970 proteins were successfully identified from quadruplicate measurements; this is the best result yet reported without the use of exhaustive pre-fractionation. Because separation efficiency in the 4-m long monolithic column system (8-h gradient, 26,805 peptides identified on average) was much higher than that in a 15-cm long, conventional particle-packed column system (65-min gradient, 10,183 peptides identified), ion suppression caused by co-elution of peptides was drastically reduced, resulting in a 5-fold improvement in MS responses on average. However, we did not observe dynamic range extension for the identified human peptides, whereas 78-fold extension was observed in our previous analysis of the Escherichia coli proteome (Anal. Chem., 82 (2010) 2616). This was probably because the current analytical technologies are still not adequate to allow acquisition of MS/MS spectra for detected precursor ions from highly complex human peptide mixtures, even though MS sensitivity was enhanced by the improved separation in this LC system. More efficient LC separation and faster MS/MS scanning are still needed for complete human proteome analysis.