title: In-depth analysis of a plasma or serum proteome using a 4D protein profiling method.
abstract: Comprehensive proteomic analysis of human plasma or serum has been a major strategy used to identify biomarkers that serve as indicators of disease. However, such in-depth proteomic analyses are challenging due to the complexity and extremely large dynamic range of protein concentrations in plasma. Therefore, reduction in sample complexity through multidimensional pre-fractionation strategies is critical, particularly for the detection of low-abundance proteins that have the potential to be the most specific disease biomarkers. We describe here a 4D protein profiling method that we developed for comprehensive proteomic analyses of both plasma and serum. Our method consists of abundant protein depletion coupled with separation strategies - microscale solution isoelectrofocusing and 1D SDS-PAGE - followed by reversed-phase separation of tryptic peptides prior to LC-MS/MS. Using this profiling strategy, we routinely identify a large number of proteins over nine orders of magnitude, including a substantial number of proteins at the low ng/mL or lower levels from approximately 300 μL of plasma sample.
authors.affiliation: Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA.
authors.forename: Hsin-Yao
authors.initials: HY
authors.lastname: Tang
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Beer
authors.forename: David W
authors.initials: DW
authors.lastname: Speicher
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D051379:Mice; D010455:Peptides / Q000302:isolation & purification; D010949:Plasma / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D044967:Serum / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
chemical_list: D001798:Blood Proteins; D010455:Peptides; D020543:Proteome; D014357:Trypsin