title: Intact-protein-based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids.
abstract: The substantial complexity and vast dynamic range of protein abundance in biological fluids, notably serum and plasma, present a formidable challenge for comprehensive protein analysis. Integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomics analysis of biological fluids. We have implemented an orthogonal three-dimensional intact-protein analysis system (IPAS), coupled with protein tagging and immunodepletion of abundant proteins, to quantitatively profile the human plasma proteome. Following immunodepletion, plasma proteins in each of paired samples are concentrated and labeled with a different Cy dye, before mixing. Proteins are subsequently separated in three dimensions according to their charge, hydrophobicity, and molecular mass. Differences in the abundance of resolved proteins are determined based on Cy dye ratios. We have applied this strategy to profile the plasma proteome for changes that occur with acute graft-versus-host disease (GVHD), following allogeneic bone marrow transplantation (BMT). Using capillary HPLC ESI Q-TOF MS, we identified 75 proteins in the micromolar to femtomolar range that exhibited quantitative differences between the pre- and post-GVHD samples. These proteins included serum amyloid A, apolipoproteins A-I/A-IV, and complement C3 that are well-known acute-phase reactants likely reflecting the post-BMT inflammatory state. In addition, we identified some potentially interesting immunologically relevant molecules including vitamin D-binding protein, fetuin, vitronectin, proline-rich protein 3 and 4, integrin-alpha, and leukocyte antigen CD97. IPAS provides a combination of comprehensive profiling and quantitative analysis, with a substantial dynamic range, for disease-related applications.
authors.affiliation: Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Shawn G
authors.initials: SG
authors.lastname: Clouthier
authors.forename: Vladimir
authors.initials: V
authors.lastname: Galchev
authors.forename: David E
authors.initials: DE
authors.lastname: Misek
authors.forename: Ulrich
authors.initials: U
authors.lastname: Duffner
authors.forename: Chang-Ki
authors.initials: CK
authors.lastname: Min
authors.forename: Rong
authors.initials: R
authors.lastname: Zhao
authors.forename: John
authors.initials: J
authors.lastname: Tra
authors.forename: Gilbert S
authors.initials: GS
authors.lastname: Omenn
authors.forename: James L M
authors.initials: JL
authors.lastname: Ferrara
authors.forename: Samir M
authors.initials: SM
authors.lastname: Hanash
mesh_terms: D000328:Adult; D000915:Antibody Affinity; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D016026:Bone Marrow Transplantation; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D006086:Graft vs Host Disease / Q000097:blood* / Q000628:therapy; D006801:Humans; D010949:Plasma / Q000737:chemistry / Q000276:immunology; D020543:Proteome / Q000032:analysis* / Q000276:immunology; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019172:Transplantation Conditioning
chemical_list: D001798:Blood Proteins; D020543:Proteome