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Copyright 2007 American Chemical Society) filtered with the above criteria, 1,578 unique peptides were positively identified from 460 distinct proteins of yeast proteome. 2. Therefore, increasing the sample loading capacity of trap column is important to improve the performance of proteome analyses. 11). Obviously, the flow rate of sample injection has little influence on the numbers of identified peptides and proteins even if the sample injection flow rate increases 20 times. Therefore, sample injection with ultra-high flow rate is feasible, which could improve the throughput of proteome analyses significantly.

The trap column was connected to a C18 separation column by a nanoflow switching valve and a microtee. During sample injection, the switching valve was switched to close the splitting flow and the flow through from the trap column was directed to waste. 5 solid line mode) including 10 min for sample loading and 20 min for equilibration. 5 dashed line mode). For manual injection, an open capillary filled with sample was connected between the microcross and the analytical column [39]. 1 % FA at a flow rate of ~200 nL/min (after splitting) and retained on the analytical column front.

First, tryptic digest of 1 μg yeast protein extract was automatically loaded onto the SCX monolithic column at 2 μL/min. 10). It could be seen that the efficient LC separation time window was about 90 min, which was consistent with the time range from 10 to 35 % ACN. Therefore, little sample loss was occurred during the processes of sample injection. L (m/z = 838, +2) extracted from the separation chromatograms, peak capacity of 359 could be calculated with ~90-min separation window. 9 Schematic diagram of the SCX monolithic column online sample injection system.

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