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Quantitative phosphoproteomics using acetone-based peptide labeling: method evaluation and application to a cardiac
Aruna B Wijeratne1, Janet R Manning, Jo El J Schultz
1Department of Cancer Biology and ‡Department of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine , 3125 Eden Avenue,Cincinnati, Ohio 45267, United States.
Journal of Proteome Research
|September 11, 2013
Summary
This study introduces reductive alkylation by acetone (RABA) labeling for quantitative phosphoproteomics. The method identified key phosphorylation changes linked to fibroblast growth factor-2 (FGF2)-mediated cardioprotection in mouse hearts.
Area of Science:
- Proteomics
- Biochemistry
- Molecular Biology
Background:
- Mass spectrometry (MS) is crucial for profiling protein phosphorylation in disease.
- Quantitative phosphoproteomics requires robust and cost-effective labeling techniques.
Purpose of the Study:
- To evaluate reductive alkylation by acetone (RABA) for quantitative phosphoproteomics.
- To investigate fibroblast growth factor-2 (FGF2)-mediated cardioprotection by analyzing cardiac phosphoproteomes.
Main Methods:
- Developed and validated a novel MS-based strategy using RABA peptide labeling.
- Compared phosphoproteomes from mouse hearts with and without low-molecular-weight FGF2 (LMW FGF2) expression.
- Utilized selected reaction monitoring (SRM) for targeted verification of phosphopeptides.
Main Results:
- Identified 14 significant phosphorylation changes on 10 proteins.
- Confirmed known and discovered novel downstream effectors of LMW FGF2 cardioprotective signaling.
- Verified cMyBPC/pSer-282 phosphorylation using SRM with isotope labeling.
Conclusions:
- RABA is an inexpensive and effective method for quantitative phosphoproteomics.
- This approach can identify biologically relevant phosphorylation changes in whole tissues.
- Provides new insights into LMW FGF2-mediated cardioprotection mechanisms.
