As emphasized in the above discussion of separation parameters, a major consideration for any comprehensive proteome analysis strategy is the total time required to analyze an entire proteome. Some improvements such as increased injection volume, smaller resin size, and longer column length can be implemented without affecting the total run time and therefore even modest improvements in protein coverage or number ofproteins identified are considered positive improvements. However, increasing the number of pixels per SDS gel lane, increasing the RP-LC gradient time for better peptide separation, and increasing gel separation distances will increase total proteome analysis time as well as increase the number of proteins identified. Therefore, a practical compromise between improved number of proteins detected and increased analysis time has to be achieved. We felt that a generally acceptable time frame for complete proteome analysis should be similar to the time required to perform a MudPIT analysis of the human plasma/serum proteome [12, 15]. Therefore, based upon the optimization results discussed in Section 3.2, we decided to fractionate the depleted plasma using MicroSol-IEF into seven fractions, followed by 1-DE of each fraction for a total distance of 4 cm (Fig. 1). Each gel lane is sliced into 2 mm size pixels to produce a total of 140 pixels. Following tryptic digestion, each pixel is analyzed by LC-ESI-MS/MS with an RP-LC gradient time of 75 min. However, to minimize carryover from the previous run especially with increased sample injection volume, a short blank gradient is run after each analytical run. Hence, the total RP-LC run time from sample to sample is 121 min (see Section 2.5). In total, 11.8 days will be required to complete the analysis of the 140 pixels from the plasma proteome. This compares favorably with ~9.8days to analyze the 135 SCX fractions of human serum proteome , and ~13.9days for 77 LC-ESI-MS/MS runs from two cycles of SCX-LC of the human plasma proteome .
Was this article helpful?