title: Immunoassay and antibody microarray analysis of the HUPO Plasma Proteome Project reference specimens: systematic variation between sample types and calibration of mass spectrometry data.
abstract: Four different immunoassay and antibody microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate-anticoagulated-plasma, EDTA-anticoagulated-plasma, or heparin-anticoagulated-plasma) and (2) evaluate the dependence on concentration of MS-based protein identifications from data sets using the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of antibody-based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances. Continued developments in antibody-based methods will further advance the scientific goals of the PPP.
authors.affiliation: The Van Andel Research Institute, Grand Rapids, MI 49503, USA. brian.haab@vai.org
authors.forename: Brian B
authors.initials: BB
authors.lastname: Haab
authors.forename: Bernhard H
authors.initials: BH
authors.lastname: Geierstanger
authors.forename: George
authors.initials: G
authors.lastname: Michailidis
authors.forename: Frank
authors.initials: F
authors.lastname: Vitzthum
authors.forename: Sara
authors.initials: S
authors.lastname: Forrester
authors.forename: Ryan
authors.initials: R
authors.lastname: Okon
authors.forename: Petri
authors.initials: P
authors.lastname: Saviranta
authors.forename: Achim
authors.initials: A
authors.lastname: Brinker
authors.forename: Martin
authors.initials: M
authors.lastname: Sorette
authors.forename: Lorah
authors.initials: L
authors.lastname: Perlee
authors.forename: Shubha
authors.initials: S
authors.lastname: Suresh
authors.forename: Garry
authors.initials: G
authors.lastname: Drwal
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Adkins
authors.forename: Gilbert S
authors.initials: GS
authors.lastname: Omenn
mesh_terms: D001798:Blood Proteins / Q000737:chemistry*; D001800:Blood Specimen Collection / Q000379:methods*; D002138:Calibration; D019343:Citric Acid / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D046228:Microarray Analysis; D040081:Protein Array Analysis; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D012016:Reference Values; D013048:Specimen Handling / Q000379:methods; D013223:Statistics as Topic
chemical_list: D001798:Blood Proteins; D019343:Citric Acid; D006493:Heparin; D004492:Edetic Acid