Abstract
The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspector. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. Quantitative information for more than 1559 proteins was obtained. In the ischemia group proteins involved in inflammation were significantly up-regulated. The ratio for calgranulin B in the ischemia/sham group was 1.81 +/- 0.97 (p < 0.0001), for complement C3 the ratio was 1.81 +/- 0.49 (p < 0.0001), and for myeloperoxidase the ratio was 1.30 +/- 0.32. Myeloperoxidase was only recently documented in Kupffer cells. The antioxidative proteins Cu,Zn-superoxide dismutase (1.34 +/- 0.19; p < 0.001) and catalase (1.23 +/- 0.43; p < 0.001) were also elevated. In conclusion, ischemia/reperfusion injury induces alterations in the Kupffer cell proteome. Isotope ratio mass spectrometry is a powerful tool to investigate these reactions. The ability to simultaneously monitor several pathways involved in reperfusion stress may result in important mechanistic insight and possibly new treatment options.
Highlights
The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells
To our knowledge there have been no studies to date that have used a proteomic approach to study the effects of warm ischemia on protein expression in Kupffer cells
The present study demonstrated that the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic approach is able to monitor several different proteins under physiological and pathophysiological conditions
Summary
The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. The activation of Kupffer cells that occurs during ischemia results in the production of oxygen radicals and the modulation of proinflammatory and anti-inflammatory cytokines [2]. These mediators are implicated in hepatic cell injury and sinusoidal function. Isotope ratio mass spectrometry with isobaric tags for relative and absolute quantitation (iTRAQ) allows for the quantification and identification of the protein content of up to four samples in one experiment by labeling all peptides in a given sample [14]. The abbreviations used are: iTRAQ, isobaric tags for relative and absolute quantitation; AIF-1, allograft inflammatory factor-1; C3, complement factor 3; C3a, activated C3; FDR, false discovery rate; IL, interleukin
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