We report the optimization of a common LC-MS/MS platform to maximize the number of proteins identified from a complex biological sample. The platform uses digested yeast lysate on a 75 microm internal diameter x 12 cm reverse-phase column that is combined with an LTQ-Orbitrap mass spectrometer. We first generated a yeast peptide mix that was quantified by multiple methods including the strategy of stable isotope labeling with amino acids in cell culture (SILAC). The peptide mix was analyzed on a highly reproducible, automated nanoLC-MS/MS system with systematic adjustment of loading amount, flow rate, elution gradient range and length. Interestingly, the column was found to be almost saturated by loading approximately 1 microg of the sample. Whereas the optimal flow rate ( approximately 0.2 microL/min) and elution buffer range (13-32% of acetonitrile) appeared to be independent of the loading amount, the best gradient length varied according to the amount of samples: 160 min for 1 microg of the peptide mix, but 40 min for 10 ng of the same sample. The effect of these parameters on elution peptide peak width is evaluated. After full optimization, 1012 proteins (clustered in 806 groups) with an estimated protein false discovery rate of approximately 3% were identified in 1 microg of yeast lysate in a single 160-min LC-MS/MS run.