Low molecular weight (LMW) proteins, protein fragments and peptides in the peripheral circulation can serve as potential clinical diagnostic makers, however the coverage of the mass spectrometry-based plasma or serum LMW proteomic analysis is limited. We found that different high concentrations of acetonitrile and 10% trichloroacetic acid (TCA) with or without DTT in the plasma extracts resulted in identification of distinct subsets of peptides in the proteomic analyses. Here we used 60% and 80% acetonitrile and 10% TCA in the absence or presence of DTT respectively to extract the plasma, and combined the six extracted samples, followed by basic pH RPLC fractionation for in-depth proteomics analysis. This approach led to profiling of 5890 peptides and 820 proteins (including those usually present in plasma at low levels), significantly expanding the current plasma LMW proteome coverage for potential clinical biomarker discovery.