Quantitative Proteomics Using 15N SILAC Mouse

Abstract

In biomedical research the use of mammalian tissues is crucial to increase our understanding of complex human diseases. Mass spectrometry-based proteomic approach has become the most powerful tool of studying large-scale protein expression profiles in mammalian tissues. To perform global proteome analysis quantification of mammalian tissues, we generated 15 N SILAC mice to obtain tissue-matched labeled peptide libraries for mass spectrometry-based quantitative proteomic analysis. We developed a new labeling protocol to circumvent adverse effects of introducing 15 N labeled diet to mice, and showed that the new labeling scheme has no significant effect on the fertility and reproduction of C57/BL6 mice. Using labeled tissues from these mice, we compared the reproducibility of mass spectrometry-based quantification with or without 15 N labeled internal standards among biological replicates of young and old brains. We found that labeled-based quantification is less susceptible to variations from instrument conditions and produces more consistent quantifications among biological replicates than label-free quantification. Lastly, we showed that over 60% of peptides from the human brain are quantifiable with internal standards from 15 N labeled mouse brain and therefore present a promising alternative of quantifying human tissues that do not have existing cell lines available for SILAC labeling. RUNNING TITLE: The utility of 15 N SILAC mouse tissues for quantitative proteomic studies