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We successfully identified the proteome expressed in Escherichia coli (E. coli) cells on a microarray scale using one-dimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a 350 cm long, 100 [micro]m i. d., monolithic silica-[C.sub.18] capillary column. E. coli tryptic digest (4 [micro]g) was injected onto the column, and a 41 h gradient was applied with a flow rate of 500 nL/min at less than 20 MPa. In total, 22 196 nonredundant tryptic peptides from 2602 proteins, including 830 membrane proteins, were identified from the E. coli cells (triplicate analysis), in which an equivalent number of genes was detected by transcriptome analysis. Approximately a 5-fold larger peak response on average was obtained in this system, compared with that obtained by conventional capillary LC-MS/MS analysis with a 15 cm long, 3 [micro]m diameter [C.sub.18] silica particle-packed column. The higher response suggests that the influence of ionization suppression was drastically reduced by the high-efficiency separation on the long monolithic silica column coupled with the shallow gradient. Because this high-resolution system does not require any additional separation prior to LC-MS/MS, this 'one-shot' proteomics approach can simplify the workflow of shotgun proteomics and minimize the sample amount, as well as reduce the total analysis time, despite the use of prolonged shallow gradient elution. 10.1021/ac100343q

Long monolithic silica-[C.sub.18] capillary columns of 100 [micro]m i.d. were prepared, and the efficiency was examined using reversed-phase HPLC under a pressure of up to 47 MPa. At linear velocities of 1-2 mm/s, 100000-500000 theoretical plates could be generated with a single column (90-440 cm in length) using an acetonitrile--water (80/ 20) mobile phase with a column dead time ([t.sub.0]) of 5-40 min. It was possible to prepare columns with a minimum plate height of 8.5 [+ or -]0.5 [micro]m and permeability of (1.45 [+ or -] 0.09) x [10.sup.-13] [m.sup.2]. The chromatographic performance of a long octadecylsilylated monolithic silica capillary column was demonstrated by the high-efficiency separations of aromatic hydrocarbons, benzene derivatives, and a protein digest. The efficiency for a peptide was maintained for an injection of up to 0.5-2 ng. When three 100 [micro]m i.d. columns were connected to form a 1130-1240 cm column system, 1000000 theoretical plates were generated for aromatic hydrocarbons with retention factors of up to 2.4 with a to of 150 min. The fact that very high efficiencies were obtained for the retained solutes suggests the practical utility of these long monolithic silica capillary columns.

Theoretical calculations are presented to optimize modulation period for maximum total peak capacity in comprehensive two-dimensional HPLC (2D-HPLC) taking into account the effect of modulation on the apparent peak capacity of the first-dimension ([sup.1]D) separation. Results indicate that modulation periods are most favorable when they are adjusted to ~2.2-4 times the standard deviation of a [sup.1]D peak in order to avoid excessively short run times at the second dimension ([sup.2]D). Data are presented that effective peak capacities of several thousand in 60 min can be expected for practical 2D-HPLC conditions, utilizing 1D gradient elution followed by [sup.2]D isocratic elution, that remain at ~50-70% of the theoretical maximum peak capacity. This work suggests that lower modulation frequencies and longer [sup.2]D separation times than previously proposed are favorable under realistic chromatographic conditions, alleviating some practical problems associated with 2D-HPLC.

Monolithic silica capillary columns for HPLC were prepared from tetramethoxysilane to have smaller sized domains and increased phase ratios as compared to previous materials, and their performance was evaluated. The monolithic silica columns possessed an external porosity of 0.65-0.76 and a total porosity of 0.92-0.95 and showed considerably higher performance and greater retention factors in a reversed-phase mode after chemical modification than columns previously reported. An octadecyisilylated monolithic silica column with the smallest domain size (through-pores of ~1.3 [micro]m and silica skeletons of ~0.9 [micro]m) showed a plate height of less than 5 [micro]m at optimum linear velocities (u) of 2-3 mm/s in 80% acetonitrile for a solute having retention factors of ~1, and ~7 [micro]m at u = 8 mm/s. With a permeability similar to that of a column packed with 5-[micro]m particles, the monolithic silica columns were able to attain column efficiencies comparable to that of particulate columns packed with 2-2.5-[micro]m particles, and showed performance in the 'forbidden region' for the previous columns. The performance of the monolithic column can be compared favorably with that of a particle-packed column when 15 000-30 000 or more theoretical plates are desired at a pressure drop of 20-40 MPa or lower. The increased homogeneity of the co-continuous structures, in addition to the small-sized domains, contributed to the higher performance as compared to previous monolithic silica columns.

Simple and comprehensive two-dimensional (2D)-HPLC was studied in a reversed-phase mode using monolithic silica columns for second-dimension (2nd-D) separation. Every fraction from the first column, 15 em long (4.6-mm i.d.), packed with fluoroalkylsilyl-bonded (FR) silica particles, was subjected to the separation in the 2nd-D using one or two octadecylsilylated ([C.sub.18]) monolithic silica columns (4.6-mm i.d., 3 cm). Monolithic silica columns in the 2nd-D were eluted at a flow rate of up to 10 mL/ min with separation time of 30 s that meets the fractionation every 15-30 s at the first dimension (1st-D) operated at a flow rate of 0.4 0.8 mL/min. Three cases were studied. (1) In the simplest scheme of 2D-HPLC, effluent of the 1st-D was directly loaded into an injector loop of 2nd-D HPLC for 28 s, and 2 s was allowed for injection. (2) Two six-port valves each having a sample loop were used to hold the effluent of the 1st-D alternately for 30 s for one 2nd-D column to effect comprehensive 2D-HPLC without the loss of 1st-D effluent. (3) Two monolithic silica columns were used for 2nd-D by using a switching valve and two sets of 2nd-D chromatographs separating each fraction of the 1st-D effluent with the two 2nd-D columns alternately. In this case, two columns of the same stationary phase ([C.sub.18]) or different phases, [C.sub.18] and (pentabromobenzyloxy)propylsilyl-bonded (PBB), could be employed at the 2nd-D, although the latter needed two complementary runs. The systems produced peak capacity of ~1000 in ~60 min in cases 1 and 2 and in ~30 min in case 3. The three stationary phases, FR, [C.sub.18], and PBB, showed widely different selectivity from each other, making 2D separations possible. The simple and comprehensive 2D-HPLC utilizes the stability and high efficiency at high linear velocities of monolithic silica columns.