Variation in the monocyte proteome.
We have determined the variability of the monocyte proteome and identified those proteins that demonstrate the greatest variation in the general control population. Monocytes were isolated from 18 healthy (9 male and 9 female) donors ages 18-50 and with no known genetic or blood disorder. A combination of Ficoll-Paque PLUS density centrifugation of cells found in the buffy coat and positive selection with monoclonal antibodies against CD14, coupled to magnetic beads, led to >98% purity of monocytes. A 100,000 g microsomal membrane fraction or 100,000 g supernatant fraction from a control subject was compared to the equivalent fractions from a distinct control subject by two-dimensional differential gel electrophoresis (2D DIGE). Those protein spots that demonstrated Cy3-/Cy5- ratios greater than 2.5-fold in at least one experiment were selected for further statistical analysis. We determined variability for 31 cytosolic and 12 membrane protein spots. Proteins have been identified for 27 of the cytosolic protein spots and 9 of the microsomal membrane protein spots by in-gel digestion with trypsin followed by reverse-phase high-performance liquid chromatography in line with tandem mass spectrometry. We identified 24 distinct monocyte proteins that demonstrated the greatest variability in this general control population. The proteins demonstrating the greatest variance in the cytosolic fraction were enolase-1 and WD (tryptophan-aspartate) repeat-containing protein 1, and in the membrane fraction they were lamin B1 and L-plastin. This study demonstrates the importance of considering variance in the control population when performing future protein profiling comparisons of monocytes derived from disease versus control populations.
Hryniewicz-Jankowska
Anita
A
Institute of Biomedical Sciences and Technology, The University of Texas at Dallas, Richardson, TX 75083, USA.
Choudhary
Pankaj K
PK
Goodman
Steven R
SR
D000293:Adolescent; D000328:Adult; D001798:Blood Proteins / Q000737:chemistry*; D002469:Cell Separation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D008280:Magnetics; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D009000:Monocytes / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
D001798:Blood Proteins