Proteomic profiling of lipopolysaccharide-activated macrophages by isotope coded affinity tagging.
Lipopolysaccharide (LPS), a glycolipid component of the outer membranes of Gram-negative bacteria, initiates proinflammatory, proapoptotic, and antiapoptotic pathways upon binding to macrophage TLR4. Macrophages that are exposed to LPS become activated and exhibit altered morphology and response to infection. We performed isotope coded affinity tagging (ICAT), multidimensional liquid chromatography, and mass spectrometry to identify proteins that are differently expressed between naive and LPS-activated macrophages. We performed replicate ICAT analyses on RAW 264.7 cultured mouse macrophages as well as C57BL/6 bone marrow derived mouse macrophages. We identified and obtained relative abundances for 1064 proteins, of which we identified 36 as having significantly different expression levels upon activation by LPS. We also compared our results with a two color microarray gene expression assay performed by the Institute for Systems Biology and observed approximately 75% agreement between mRNA transcription and protein expression regarding up- or down-regulation of gene products. We used Western blot analysis to confirm the findings of ICAT and mRNA for one protein, sequestosome 1, the cellular concentration of which was observed to increase upon activation by LPS.
Swearingen
Kristian E
KE
Department of Chemistry, University of Washington, P.O. Box 351700, Seattle, Washington 98195-1700, USA.
Loomis
Wendy P
WP
Zheng
Meng
M
Cookson
Brad T
BT
Dovichi
Norman J
NJ
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D006360:Heat-Shock Proteins / Q000032:analysis / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D040081:Protein Array Analysis; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D000071456:Sequestosome-1 Protein
D048868:Adaptor Proteins, Signal Transducing; D006360:Heat-Shock Proteins; D008070:Lipopolysaccharides; D020543:Proteome; D000071456:Sequestosome-1 Protein; C101343:Sqstm1 protein, mouse