Proteome signatures of inflammatory activated primary human peripheral blood mononuclear cells

Abstract

Proteome profiling is the method of choice to identify marker proteins whose expression may be characteristic for certain diseases. The formation of such marker proteins results from disease-related pathophysiologic processes. In healthy individuals, peripheral blood mononuclear cells (PBMCs) circulate in a quiescent cell state monitoring potential immune-relevant events, but have the competence to respond quickly and efficiently in an inflammatory manner to any invasion of potential pathogens. Activation of these cells is most plausibly accompanied by characteristic proteome alterations. Therefore we investigated untreated and inflammatory activated primary human PBMCs by proteome profiling using a ‘top down’ 2D-PAGE approach in addition to a ‘bottom up’ LC–MS/MS-based shotgun approach. Furthermore, we purified primary human T-cells and monocytes and activated them separately. Comparative analysis allowed us to characterize a robust proteome signature including NAMPT and PAI2 which indicates the activation of PBMCs. The T-cell specific inflammation signature included IRF-4, GBP1and the previously uncharacterized translation product of GBP5; the corresponding monocyte signature included PDCD5, IL1RN and IL1B. The involvement of inflammatory activated PBMCs in certain diseases as well as the responsiveness of these cells to anti-inflammatory drugs may be evaluated by quantification of these marker proteins.This article is part of a Special Issue entitled: Integrated omics.

Coffee consumption modulates inflammatory processes in an individual fashion

“…The technical feasibility of this approach was investigated by 2D‐PAGE analysis of metabolically labeled PBMCs. In this setting, a marked upregulation of several cytoplasmic marker molecules described previously was detectable upon inflammatory stimulation (Fig. A).…”

“…However, a quantitative assessment of an inflammation status is not trivial. We have demonstrated previously, that metabolic labeling of PBMCs with 35 S‐methionine/cysteine and subsequent 2D‐PAGE analysis may allow such evaluation . Applying this method, we observed rather subtle, but still unexpected effects of coffee consumption (Fig.…”

Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

“…Cox-1 (Q63921), an important mediator of inflammatory signalling, was observed induced by PB in stroma cells in one animal only, which may be due to low protein concentration at the limit of detection. The expression of the inflammation-related protein MX-1 [58] in the control stroma cells, however, indicates that the presently employed cell manipulation steps may also have altered the inflammatory activity state in an artificial way, rendering clear conclusions, with respect to inflammatory pathways, difficult. The present observations still indicate some complex regulatory actions of PB which are related to but still distinct from classical inflammatory activation warranting further investigation.…”

Proteome profiling of keratinocytes transforming to malignancy

“…To generate a subproteome that helps to collect more biologically relevant information , we performed subcellular fractionation and analyzed the environmental influence by stimulation with IL‐1beta. Subcellular fractionation was performed as described previously . Cytoplasmic, nuclear, and secreted proteins were isolated from keratinocytes and A431 cells lysed in 10 mM HEPES/NaOH, pH 7.4, 0.25 M sucrose, 10 mM NaCl, 3 mM MgCl 2 , and 0.5% Triton X‐100, supplemented with protease inhibitors (1 mM PMSF, and aprotinin, leupeptin, and pepstatin A; each 1 μg/mL).…”