Systematic comparison of fractionation methods for in-depth analysis of plasma proteomes

Discovery and validation of plasma biomarkers are quite challenging because of the high complexity and wide dynamic range of the plasma proteome. Current plasma protein profiling strategies usually use major protein immunodepletion and nanoLC-MS/MS as the first and final analytical steps, respectively, but additional fractionation is needed to detect and quantify low-abundance disease biomarkers. In this study, the performances of 1-D SDS-PAGE, peptide isoelectrofocusing, and peptide high pH reverse-phase chromatography for fractionation of immunodepleted human plasma were systematically compared by evaluating protein coverage, peptide resolution, and capacity to detect known low-abundance proteins. Trade-offs between increasing the number of fractions to improve proteome coverage and resulting decreases in throughput also were assessed. High pH reverse-phase HPLC exhibited the highest peptide resolution and yielded the best depth of analysis with detection of the largest number of known low-abundance proteins for a given level of fractionation. Another advantage of using high pH reverse-phase fractionation rather than 1-D SDS gels is that all fractionation steps except for abundant protein depletion occur at the peptide level, making this strategy more compatible with quantitative biomarker validation methods such as stable isotope dilution multiple reaction monitoring.