File S1 - Modifications in Rat Plasma Proteome after Remote Ischemic Preconditioning (RIPC) Stimulus: Identification by a SELDI-TOF-MS Approach

Figure S1. Profiling of the proteins corresponding to the m/z peaks. Representative SELDI-TOF-MS protein spectra of plasma sample from one Control, one RIPC 5′, and one RIPC 10′ rat for the 13720, 27340, 42463–42427, 42610 and 54700 m/z peaks. Results are presented as intensities of SELDI-TOF reading (arbitrary units). The 27340, 42427 and 42610 m/z peaks were found to be differentially expressed on the H50 array and the 13720, 42463 and 54700 m/z peaks on the CM10 array. The statistical significance was calculated by the Mann-Whitney test. Figure S2. Quantification of the proteins corresponding to the m/z peaks. Scattergrams showing the significant differences in intensity of each peaks 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). Detailed p-value data for comparison between the three groups is indicated in Table 2. Figure S3. Purification by liquid-phase electrofocusing. Protein corresponding to the m/z peaks indicated were purified using the MicroRotofor® cell. SELDI-TOF-MS protein spectra analysis of fractions from pH gradient 7–9 for 13720, 42463–42427 and 42610 m/z peaks, pH gradient 3–10 for 27340 m/z peak and 5–7 for 54700 m/z peak. Figure S4. Purification by gel electrophoresis. Each fraction obtained by liquid-phase electrofocusing was analyzed on NU-PAGE 10% coomassie blue stained-gel. The band corresponding to the peak of interest was framed. Figure S5. Identification of the proteins corresponding to the m/z peaks. Identification of the m/z peaks purifed by gel electrophoresis by mass spectrometry. Aminoacids indicated in red corresponds to the peptides identified in the protein sequence. Figure S6. Identification of the proteins corresponding to the m/z peaks. SELDI-TOF-MS protein spectra of crude (untreated) and immunodepleted plasmas with antibodies (depleted) showed the decrease in the corresponding m/z peak following immunodepletion, validating the identification. Figure S7. Profiling and verification of identified proteins corresponding to the 9420 and 15870–15980 m/z peaks. 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). B: The 9420 and 15870–15980 m/z peak were respectively found to be differentially expressed on the H50 and CM10 arrays as calculated by the Mann-Whitney test. Scattergram showing the significant differences in intensity of 9420 (left panel), 15870 (middle panel) and 15980 (right panel) m/z peaks 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). Detailed p-value data for comparison between the three groups is indicated in Table 2. C: SELDI-TOF-MS protein spectra of crude (untreated) and immunodepleted plasma (10 µL) with 10 µg of haptoglobin or 5 µg of hemoglobin antibody (depleted) showed the decrease in 9420 and 15870–15980 m/z peak following immunodepletion, validating the identifications. (DOCX)