title: Systematic comparison of fractionation methods for in-depth analysis of plasma proteomes.
abstract: 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.
authors.affiliation: Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute , Philadelphia, Pennsylvania, United States.
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Cao
authors.forename: Hsin-Yao
authors.initials: HY
authors.lastname: Tang
authors.forename: Huan
authors.initials: H
authors.lastname: Wang
authors.forename: Qin
authors.initials: Q
authors.lastname: Liu
authors.forename: David W
authors.initials: DW
authors.lastname: Speicher
mesh_terms: D015415:Biomarkers / Q000097:blood; D001774:Blood Chemical Analysis; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D007525:Isoelectric Focusing; D010446:Peptide Fragments / Q000302:isolation & purification; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D015415:Biomarkers; D001798:Blood Proteins; D010446:Peptide Fragments; D020543:Proteome