Two-dimensional gel electrophoresis of synovial fluid: method for detecting candidate protein markers for osteoarthritis.
Synovial fluid (SF) is a dynamic reservoir for proteins originating from serum, synovial tissue, and cartilage. The composition of the SF proteome may reflect the pathophysiological conditions affecting the circulatory system and cartilage. Our long-term goal is to identify reliable protein markers for osteoarthritis (OA) in SF. We first evaluated the pattern of SF proteins on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as a function of protein loading, pH range for isoelectric focusing, and concentration of acrylamide in SDS-PAGE. Removal of albumin and Gamma-globulins from the samples did not improve the detection of protein spots on 2D-PAGE. The repeatability of protein spot intensity was tested by triplicate 2D-PAGE of a given sample; these experiments showed low intrasample variability (correlation coefficients 0.89-0.95). Differences between multiple samples were tested by comparing the 2D-PAGE of four samples. These experiments showed slightly greater variation between samples (correlation coefficients 0.85-0.93) and a number of differentially expressed proteins. The intensity of 18 protein spots differed more than fivefold, and the intensity of nine protein spots differed more than 100-fold. These results show that 2D-PAGE can be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA.
Yamagiwa
Hiroshi
H
Department of Orthopedic Surgery, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA.
Sarkar
Gobinda
G
Charlesworth
M Cristine
MC
McCormick
Daniel J
DJ
Bolander
Mark E
ME
D015415:Biomarkers / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods; D020370:Osteoarthritis, Knee / Q000378:metabolism*; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D013582:Synovial Fluid / Q000737:chemistry*
D015415:Biomarkers; D011506:Proteins