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A typical goal of many bottom-up proteomics experiments is to characterize as many proteins as possible over a wide dynamic range. Latest trends in proteomics point to the need to analyze multiple biological and technical replicates in order to confidently identify proteins or measure statistically significant changes in quantitative experiments. One of the bottlenecks in running nanoflow LC/MS of proteomics samples is the traditional long gradient length (two hours or more). Additionally, sample loading time and gradient delay in which no peptides elute from the column at low flow rates also limit sample throughput. In this study, LC conditions will be optimized to maximize chromatographic resolution and sample throughput to identify and quantify as many proteins as possible per day. Key to this work is a new column chemistry (1.8 μm HSS T3), which is more retentive than the packing material used in the trapping column (5 μm Symmetry C18) and even more retentive than the analytical column material currently used (1.7 μm bridged ethyl hybrid (BEH)). This new chemistry allows for better re-focusing of the peptides on the analytical column after trapping, yielding narrower peak widths and better peak capacity. With the T3 column, higher column temperatures can be used without sacrificing peptide focusing, so higher flow rates can be used to increase throughput and resolution without increasing pressure on the system. The reduction in gradient delay due to higher flow rate, along with optimized loading, trapping, and re-equilibration conditions, means that peptides elute over the majority of the chromatogram. Data from multiple sample types, including different rat organs, will be shown. Two-dimensional chromatography using high-low pH RP/RP will also be evaluated using the optimized method for the second dimension separation.