Modifications in rat plasma proteome after remote ischemic preconditioning (RIPC) stimulus: identification by a SELDI-TOF-MS approach

Abstract

Remote ischemic preconditioning's (RIPC) ability to render the myocardium resistant to subsequent prolonged ischemia is now clearly established in different species, including humans. Strong evidence suggests that circulating humoral mediators play a key role in signal transduction, but their identities still need to be established. Our study sought to identify potential circulating RIPC mediators using a proteomic approach. Rats were exposed to 10-min limb ischemia followed by 5- (RIPC 5') or 10-min (RIPC 10') reperfusion prior to blood sampling. The control group only underwent blood sampling. Plasma samples were isolated for proteomic analysis using surface-enhanced laser desorption and ionization - time of flight - mass spectrometry (SELDI-TOF-MS). A total of seven proteins, including haptoglobin and transthyretin, were detected as up- or down-regulated in response to RIPC. These proteins had previously been identified as associated with organ protection, anti-inflammation, and various cellular and molecular responses to ischemia. In conclusion, this study indicates that RIPC results in significant modulations of plasma proteome.

Figure 1. Venn diagram demonstrating the number of SELDI peaks detected and analysed for the comparison between the CRTL, RIPC 5′ and RIPC 10′ groups.

A: Comparison of the number of peaks detected before or after CPLL treatment of plasma samples in CM10 and H50 arrays. B: Comparison of the number of peaks with significant modulations of intensity in the CRTL, RIPC 5′ and RIPC 10′. All the samples were analyzed in duplicate.

Figure 2. Profiling, purification and identification of the protein corresponding to the 8317 m/z peak.

A: Representative SELDI-TOF-MS protein spectra of plasma sample from one Control, one RIPC 5′, and one RIPC 10′ rat. Results are presented as intensities of SELDI-TOF reading (arbitrary units). The 8317 m/z peak was found to be differentially expressed on the CM10 array as calculated by the Mann-Whitney test (left panel). Scattergram showing the significant differences in intensity of 8317 m/z peak in plasma samples derived from Control, RIPC 5′, and RIPC 10′ rats. The continuous line represents the mean, and dots represent each individual rat (n = 10 in each group) (right panel). Detailed p-value data for comparison between the three groups is indicated in Table 2. B: Purification of the protein corresponding to the 8317 m/z peak. SELDI-TOF-MS protein spectrum of fractions F1, F2, F3, F4, F5 and F6 from MicroRotofor® cell, by a pH gradient 7–9 (left panel). Each fraction (F1, F2, F3, F4, F5 and F6) was analyzed on NU-PAGE 10% coomassie blue stained-gel. The band corresponding to the 8317 m/z peak was framed (right panel). C: Identification of the 8317 m/z peak by mass spectrometry. Aminoacids indicated in red corresponds to the peptides identified in Apolipoprotein C-III (ApoC-III) (left panel). SELDI-TOF-MS protein spectra of crude (untreated) and immunodepleted plasma (20 µL) with 10 µg ApoC-III antibody (depleted) showed the decrease in 8317 m/z peak following immunodepletion, validating the identification (right panel).