Recent advances in chromatography and mass spectrometry (MS) have made rapid and deep proteomic profiling possible. To maximize the performance of the recently produced Orbitrap hybrid mass spectrometer, we have developed a protocol that combines improved sample preparation (including optimized cellular lysis by extensive bead beating) and chromatographic conditions (specifically, 30-cm capillary columns packed with 1.7-μm bridged ethylene hybrid material) and the manufacture of a column heater (to accommodate flow rates of 350-375 nl/min) that increases the number of proteins identified across a single liquid chromatography-tandem MS (LC-MS/MS) separation, thereby reducing the need for extensive sample fractionation. This strategy allowed the identification of up to 4,002 proteins (at a 1% false discovery rate (FDR)) in yeast (Saccharomyces cerevisiae strain BY4741) over 70 min of LC-MS/MS analysis. Quintuplicate analysis of technical replicates reveals 83% overlap at the protein level, thus demonstrating the reproducibility of this procedure. This protocol, which includes cell lysis, overnight tryptic digestion, sample analysis and database searching, takes ∼24 h to complete. Aspects of this protocol, including chromatographic separation and instrument parameters, can be adapted for the optimal analysis of other organisms.