title: The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins.
abstract: Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.
authors.affiliation: Large Scale Biology Corporation, 20451 Seneca Meadows Parkway, Germantown, MD 20876, USA. rembert.pieper@lsbc.com
authors.forename: Rembert
authors.initials: R
authors.lastname: Pieper
authors.forename: Christine L
authors.initials: CL
authors.lastname: Gatlin
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Makusky
authors.forename: Paul S
authors.initials: PS
authors.lastname: Russo
authors.forename: Courtney R
authors.initials: CR
authors.lastname: Schatz
authors.forename: Stanton S
authors.initials: SS
authors.lastname: Miller
authors.forename: Qin
authors.initials: Q
authors.lastname: Su
authors.forename: Andrew M
authors.initials: AM
authors.lastname: McGrath
authors.forename: Marla A
authors.initials: MA
authors.lastname: Estock
authors.forename: Prashanth P
authors.initials: PP
authors.lastname: Parmar
authors.forename: Ming
authors.initials: M
authors.lastname: Zhao
authors.forename: Shih-Ting
authors.initials: ST
authors.lastname: Huang
authors.forename: Jeff
authors.initials: J
authors.lastname: Zhou
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.forename: Ricardo
authors.initials: R
authors.lastname: Esquer-Blasco
authors.forename: N Leigh
authors.initials: NL
authors.lastname: Anderson
authors.forename: John
authors.initials: J
authors.lastname: Taylor
authors.forename: Sandra
authors.initials: S
authors.lastname: Steiner
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001798:Blood Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014466:Ultraviolet Rays
chemical_list: D001798:Blood Proteins; D020543:Proteome