%0 Journal Article
%T Quantitative Proteomics Using 15N SILAC Mouse - Quantitative Proteomics Using 15N SILAC Mouse - Open Access Pub
%A Antonius Koller
%A Emily I. Chen
%A Holly Colognato
%A Jenne Relucio
%A Ruofeng Wen
%A Xiao Wu
%J OAP | Home | Journal of Proteomics and Genomics Research | Open Access Pub
%D 2018
%X 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 15N 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 15N 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 15N 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 15N labeled mouse brain and therefore present a promising alternative of quantifying human tissues that do not have existing cell lines available for SILAC labeling. DOI10.14302/issn.2326-0793.jpgr-13-252 Liquid chromatography (LC) coupled to electrospray mass spectrometry (MS) is well established in shotgun proteomics to rapidly identify and quantify large numbers of proteins. Quantification of identified proteins from different samples in LC-MS/MS is performed using certain physical attributes of peptides as surrogate measurements. One approach to perform global quantitative proteome comparisons by LC-MS/MS is to include a labeled version of peptides in the samples. Stable-isotope labeled amino acids or isobaric chemical mass tags can be introduced to cells through the growth media (SILAC)1, 2, 3 or covalently linked to proteolyzed peptides (ICAT, iTRAQ, or TMT)4, 5, 6, 7, 8, 9 respectively to generate internal standards for LC-MS/MS based quantification. Alternatively, label-free LC-MS/MS based protein quantification can be achieved by measuring mass spectral peak intensities of peptide ions or the total number of spectra identified for a protein (spectral counting)10, 11, 12. Although label-based protein quantification methods are superior for quantifying biological relevant changes, label-free protein quantification methods have
%U https://www.openaccesspub.org/jpgr/article/45