A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system.
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.
Misek
David E
DE
Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, MI, USA.
Kuick
Rork
R
Wang
Hong
H
Galchev
Vladimir
V
Deng
Bin
B
Zhao
Rong
R
Tra
John
J
Pisano
Michael R
MR
Amunugama
Ravi
R
Allen
David
D
Walker
Angela K
AK
Strahler
John R
JR
Andrews
Philip
P
Omenn
Gilbert S
GS
Hanash
Samir M
SM
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*
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