Cardiac phosphoproteomics during remote ischemic preconditioning: a role for the sarcomeric Z-disk proteins

Abstract

Remote ischemic preconditioning (RIPC) induced by brief ischemia/reperfusion cycles of remote organ (e.g., limb) is cardioprotective. The myocardial cellular changes during RIPC responsible for this phenomenon are not currently known. The aim of this work was to identify the activation by phosphorylation of cardiac proteins following RIPC. To achieve our aim we used isobaric tandem mass tagging (TMT) and reverse phase nanoliquid chromatography tandem spectrometry using a Linear Trap Quadropole (LTQ) Orbitrap Velos mass spectrometer. Male C57/Bl6 mice were anesthetized by an intraperitoneal injection of Tribromoethanol. A cuff was placed around the hind limb and inflated at 200 mmHg to prevent blood flow as confirmed by Laser Doppler Flowmetry. RIPC was induced by 4 cycles of 5 min of limb ischemia followed by 5 min of reperfusion. Hearts were extracted for phosphoproteomics. We identified approximately 30 phosphoproteins that were differentially expressed in response to RIPC protocol. The levels of several phosphoproteins in the Z-disk of the sarcomere including phospho-myozenin-2 were significantly higher than control. This study describes and validates a novel approach to monitor the changes in the cardiac phosphoproteome following the cardioprotective intervention of RIPC and prior to index ischemia. The increased level of phosphorylated sarcomeric proteins suggests they may have a role in cardiac signaling during RIPC.

Figure 1

Phospho-TMT workflow. (a) Structure of the TMT Tag. Six tags are available, each with a different reporter group (m/z 126, 127, 128, 129, 130, or 131). The mass normaliser region balances out the difference in mass in the reporter groups such that the overall tag mass is constant. The reactive group provides amine-specific labelling. (b) Six samples are digested with trypsin to generate peptides which are then labelled with one of the six TMT tags. The labelling reaction is quenched and the samples are pooled. The pooled sample is passed through a titanium dioxide (TiO₂) column, phosphopeptides bind to the column while nonphosphorylated peptides pass straight through. The phosphopeptides are then eluted and analysed by LC-MS/MS. Differentially tagged peptides are indistinguishable at the MS1 level since the overall tag mass is constant. Fragmentation of the peptides detected in the MS1 spectra produces secondary MSMS spectra for each peptide, allowing elucidation of the peptide sequence. In addition, the fragmentation process causes cleavage of the linker region within the tag, releasing the reporter groups which appear as a cluster of ions at the low mass end of each MSMS spectra. The relative intensity of the members of this ion cluster shows the relative abundance of that peptide between the six samples under comparison.

Figure 2

Quantification of phosphorylated phospholamban (p-PLB) in the same samples measured using Western blotting or TMT tandem mass tagging. Protein expression level for cardiac p-PLB (GAPDH in lower panel) in RIPC (126, 127, and 128) and control (129, 130, and 131) samples are shown in (a). Raw data for identification (c) and quantification (b) of the phosphopeptide RASTIEMPQQAR from PLB using TMT isobaric mass tagging are shown in (b) & (c). The reporter ion cluster from the six different mass tags are shown boxed in panel (c) and expanded in bar chart form in panel (b) for both RIPC (numbers: 126, 127, and 128) and control (129, 130, and 131) samples. The peptide sequence showing the phosphorylated amino acid (Ser 16, underlined) is also shown in panel (b). The samples used in these measurements were taken from series 1.

Figure 3

Phosphopeptide identification and quantification for phospho-myozenin-2 from RIPC and control samples using Tandem Mass Tagging. P-myozenin-2 peptide phosphorylated at threonine 107 is shown in panel (a). The mass peaks generated by different mass tags (reporter ions) for different samples are magnified and shown in bar chart form in panel (a) for both RIPC (numbers: 126, 127, and 128) and control (129, 130, and 131) samples. The (MS/MS) spectra for the peptide used for protein identification and the reporter ions (boxed) are shown in (b). The samples used in these measurements were taken from Series 1.

Figure 4

Total myozenin-2 in ventricular tissues extracted from RIPC (n = 5) and control (n = 4) samples. Upper panel is a representative blot showing myozenin-2 and GAPDH. The graph shows individual myozenin-2 levels (normalized to GAPDH) as well as the mean ± SEM for each intervention. There was no statistical difference between the two groups.