An Acetone-based Peptide Labeling and Mass Spectrometry Phosphoproteomics Workflow Enables Identification of Biomolecular Targets Relevant to a Fibroblast Growth Factor Induced Post-ischemic Cardiac Recovery

摘要

Protein phosphorylation modulates normal cellular functions, and disease initiations/progressions, thus understanding phosphoproteomic changes of biological systems in response to external stimuli is essential for therapeutic interventions. Mass spectrometry (MS) methods are currently popular to study such changes, but have yet to become common in solving biological problems; partly due to expensive differential peptide tagging chemistries like iTRAQ (Isobaric Tags for Relative/Absolute Quantification) that are pivotal in comparing phosphoproteomes. A cost-effective MS-workflow that relies on acetone (d0 and d6) for peptide labeling which is amenable to phosphopeptide enrichment by TiO2-chromatography has been developed. This was achieved by applying the workflow to standard phosphoprotein alpha-casein and monitoring its predetermined quantitative ratios using mass spectrometry. Here, we apply the workflow to evaluate its robustness in delivering biologically relevant phosphoproteomic targets or elucidating signaling networks related to a cardiac injury model for subsequent therapeutic interventions. /9LMW FGF2 (low-molecular weight fibroblast growth factor 2), improves post-ischemic recovery of cardiac function, which is mediated by various protein kinase signaling cascades. However, much is unknown about downstream targets and their phosphorylation changes induced by LMW FGF2 for improved cardiac function. Thus, using the developed workflow, five biologically distinct pair wise phosphoproteomic comparisons of cardiac tissue extracts obtained from “LMW FGF2-expressed” and “not-expressed” mouse hearts subjected to 60 minutes of ischemia and 5 minutes of reperfusion were performed. Alpha-casein was also “spiked-in” into the cardiac tissue extracts as an external standard to monitor procedural errors associated with the workflow. Phosphoproteomic differences in potential targets were revealed that are previously recognized and biologically relatable to cardioprotection signaling by LMW FGF2—e.g. cardiac myosin binding protein C (cMyBP-C) phosphorylations at Ser273, Ser282 and Ser-284, and connexin-43 (Cx43) phosphorylation, respectively. Also, identified phosphoproteins were several new LMW FGF2 targets that are being further evaluated in our labs for their significance. Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities.