Large-bore particle-entrapped monolithic precolumns prepared by a sol-gel method for on-line peptides trapping and preconcentration in multidimensional liquid chromatography system for proteome analysis

摘要

The present report describes the preparation and characterization of large-bore particle-entrapped monolithic precolumns, which are suitable for incorporation into a two-dimensional liquid chromatography (2D-LC) system for proteome analysis. The fritless precolumns with different inner diameter (i.d.) (320 and 530 mu m) were rapidly and successfully prepared by entrapping octadecylsilica (ODS) particles (5 mu m, 300 X) prepacked into fused silica capillaries with a sol-gel network, which was formed by hydrolysis and polycondensation of methyltriethoxysilane (MTES). By optimizing the composition of the sol solution, the resulting large-bore monolithic precolumns of 5 turn length allow a flow rate of 20 mu L/min loading buffer at a reasonable low back pressure of 25 bar or less and are capable of withstanding up to 300 bar inlet pressure. Scanning electron micrograms of the precolumns profile showed that the evolving sol-gel network joined particles to each other and onto the column wall, and no cracking or shrinkage of the column bed was observed even in 530 mu m-i.d. capillary. The performance of the particle-entrapped monolithic precolumns used for preconcentration and desalting of proteolytic digest was evaluated by on-line coupling the large-bore precolumns with a capillary reversed-phase liquid chromatographic (RPLC) column followed by UV detection. The laboratory-made monolithic precolumns with 320 and 530 mu m i.d. were characterized by using BSA tryptic digest or peptide standards as the analytes with respect to sample loading capacity, linearity, recovery and reproducibility, etc. The results indicate that the large-bore and short precolumns (5 mm x 320 mu m W. or 5 mm, x 530 mu m i.d.) allow sample fast loading at a flow rate of 30 or 60 mu L/min. The precolumns also have a mass loading capacity for BSA peptides of about 70 mu g and for standard peptides of about 80 mu g. Good linear calibration curves (R-2 > 0.99) were obtained and the limits of detection (signal-to-noise ratio, S/N = 3) were improved by more than 60-fold and were between 0.53 and 1.32 ng/mu L even with a UV absorbance detector. The total recovery was found to be approximately 90-100 % for BSA digest and standard peptides. The day-to-day relative standard deviation (RSD) values for recoveries of BSA peptides on a single precolumn ranged from 4.66 to 7.56 % and 2.68 to 3.05 % for precolumn back pressure, while the column-to-column RSD values were 3.51-6.13 % and 1.22-1.26 % for recoveries of BSA peptides and precolumn back pressure, respectively. With good precolumn reproducibility, no significant degradation or decrease in precolumn performance was showed even after similar to 150 preconcentration/desorption cycles. The precolumns also proved to be resistant to salt buffer with high concentration and low-pH mobile phase. The large-bore particle-entrapped monolithic precolumns will be further used in a high-throughput 2D-LC array system coupled with tandem matrix assisted laser desorption/ionization-time of fligt-time of flight-mass spectrometry (MALDI-TOF-TOF-MS) detection for proteome analysis. (c) 2005 Elsevier B.V. All rights reserved.