LC/MS identification of 12 intracellular cytoskeletal and inflammatory proteins from monocytes adherent on surface-adsorbed fibronectin-derived peptides.
The extent and duration of the host response determines device efficacy, yet the mechanism is poorly understood. U937 promonocytic cells were cultured on peptide-adsorbed tissue-culture polystyrene to better understand surface-modulated intracellular events. Phosphotyrosine proteins were enriched by immunoprecipitation and analyzed by nanospray HPLC-coupled tandem mass spectrometry (LC/MS). Tyrosine-phosphorylated proteins were chosen based on physiological significance and previous densitometry results, which identified a set of proteins ranging from approximately 200 to approximately 23 kDa showing altered phosphorylation levels in response to various surface-adsorbed ligands and phosphorylation inhibitor AG18. Although LC/MS has been used for nearly a decade, its application to the field of biomaterials is relatively novel. Twelve intracellular proteins identified by nanospray LC/MS are potentially related to the host response. Eight of the twelve proteins are related to the cytoskeleton including: moesin, heat shock protein 90beta, alpha-tubulin, elongation factor 1alpha, beta actin, vimentin, plasminogen activator inhibitor 2, and heterogeneous ribonuclear protein A2. The remaining four proteins: high mobility group box 1, caspase recruitment domain 5, glycoprotein 96, and heterogeneous nuclear ribonucleoprotein D0 modulate inflammation. The specific effect each peptide has upon modulating the phosphorylation state of these proteins cannot be determined from this work; however, 12 viable targets have been identified for further investigation into the role each plays in the surface-mediated monocyte response.
Zuckerman
Sean T
ST
Department of Biomedical Engineering, School of Pharmacy, University of Wisconsin-Madison, Wisconsin 53705, USA.
Kao
Weiyuan John
WJ
D002448:Cell Adhesion; D002853:Chromatography, Liquid; D016207:Cytokines / Q000096:biosynthesis*; D005353:Fibronectins / Q000737:chemistry*; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism*; D013058:Mass Spectrometry; D009000:Monocytes / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D020298:U937 Cells
D016207:Cytokines; D005353:Fibronectins; D018836:Inflammation Mediators; D010455:Peptides