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Multiple LC-MS strategies were applied to the analysis of extracted tryptic peptides from Formalin-fixed paraffin-embedded (FFPE) tissue archives. Five FFPE tissue samples were independently extracted, digested, and analyzed using LC-MS to assess technical reproducibility and variability in a novel sample preparation procedure. Subsequent 1D LC-MS experiments are compared to a 2D RP-RP LC-MS approach to assess depth of proteome coverage and analytical reproducibility. Samples were analysed in triplicate by 1D LC-MS, 2D-5step and 2D-10step at 500ng, 2.5μg, and 4.5μg respectively. 2D separations were performed using a discontinuous step gradient at pH=10 in the first dimension, followed by a pH=2 separation in the second dimension/ The eluent was coupled to the nanoelectrospray source of a Synapt MS, using MSE. Data was processed with PLGS2.4. The different FFPE tissue extracts showed highly consistent proteins identified across the replicates, using 1D LC-MS. The overlap was high with 80% of the proteins common across the samples, showing reproducible qualitative and quantitative metrics at the protein and peptide level. For the 2D analysis an increasing number of proteins are identified when the number of first dimension steps is increased. Approximately 250-300 proteins are identified in each of the pseudo 1D experiments, >450 proteins in each 2D 5 step experiment and >600 proteins in each 2D 10 step experiment. High reproducibility between replicates of the 2D RP/RP technique is observed at both the peptide and protein level for each individual step and for each of the steps combined. Absolute quantitation, utilizing the intensity of the top three peptides from the yeast ADH internal standard, shows that the with an increased number of first dimension steps, there is an increase in the number of low abundant species that are sampled as interferences from higher abundant species are reduced with the greater separation capability when using more steps.