Selected reaction monitoring mass spectrometry for relative quantification of proteins involved in cellular life and death processes.

Abstract

Monitoring of proteins involved in cellular life and death processes is of high scientific interest since it permits the elucidation of functional changes in a variety of diseases. In this study, we have developed a nanoLC-MS/MS assay for the simultaneous detection and quantification of 24 selected proteins that are known to be important for cellular homeostasis. The Selected Reaction Monitoring (SRM) assay applies heavy-labeled peptide analogues for the relative quantification of proteins with central functions in cellular stress and metabolism, including many mitochondrial proteins. The assay includes proteins involved in the quality control of mitochondrial proteins, oxidative stress, respiratory chain, and fatty acid degradation, as well as the cytosolic glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase and ribosomal proteins. The assay can thus quantitate the balance between mitochondrial and cytosolic pathways, which is relevant in many disease states, and can be studied by comparing patient and control samples. The measured validation parameters showed satisfactory results for the proteins included in the analysis. The linear range of the monitored proteins was 0.01-20nM, with a median precision of less than 10%. The assay performed well in monitoring proteins in both cultured human skin fibroblast cells as well as in isolated peripheral blood mononuclear cells. We therefore believe that this assay is applicable for the study of cellular stress response in various types of cell defects and disease states.