title: A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system.
abstract: We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.
authors.affiliation: Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, MI, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Misek
authors.forename: Rork
authors.initials: R
authors.lastname: Kuick
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Vladimir
authors.initials: V
authors.lastname: Galchev
authors.forename: Bin
authors.initials: B
authors.lastname: Deng
authors.forename: Rong
authors.initials: R
authors.lastname: Zhao
authors.forename: John
authors.initials: J
authors.lastname: Tra
authors.forename: Michael R
authors.initials: MR
authors.lastname: Pisano
authors.forename: Ravi
authors.initials: R
authors.lastname: Amunugama
authors.forename: David
authors.initials: D
authors.lastname: Allen
authors.forename: Angela K
authors.initials: AK
authors.lastname: Walker
authors.forename: John R
authors.initials: JR
authors.lastname: Strahler
authors.forename: Philip
authors.initials: P
authors.lastname: Andrews
authors.forename: Gilbert S
authors.initials: GS
authors.lastname: Omenn
authors.forename: Samir M
authors.initials: SM
authors.lastname: Hanash
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology; D015415:Biomarkers; D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002232:Carbocyanines / Q000494:pharmacology; D002845:Chromatography; D002853:Chromatography, Liquid; D004492:Edetic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D013058:Mass Spectrometry; D008970:Molecular Weight; D020033:Protein Isoforms / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000379:methods*
chemical_list: D000925:Anticoagulants; D015415:Biomarkers; D001798:Blood Proteins; D002232:Carbocyanines; D005456:Fluorescent Dyes; D020033:Protein Isoforms; D020543:Proteome; C089187:cyanine dye 3; C085321:cyanine dye 5; D004492:Edetic Acid