Journal
ANALYTICAL CHEMISTRY
Volume 86, Issue 5, Pages 2314-2319Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac403579s
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Funding
- National Institutes of Health [R01 GM080148, R01DK098672, DP2OD007447, 5T32GM007215-37]
- National Science Foundation [DGE-1256259]
- National Science Foundation Early Faculty Career grant from the Office of the Director
- NSF
- NIH [T32GM008505]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1262672] Funding Source: National Science Foundation
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The ability to acquire highly accurate quantitative data is an increasingly important part of any proteomics experiment, whether shotgun or top-down approaches are used. We recently developed a quantitation strategy for peptides based on neutron encoding, or NeuCode SILAC, which uses closely spaced heavy isotope-labeled amino acids and high-resolution mass spectrometry to provide quantitative data. We reasoned that the strategy would also be applicable to intact proteins and could enable robust, multiplexed quantitation for top-down experiments. We used yeast lysate labeled with either (C6N2)-C-13-N-15-lysine or H-2(8)-lysine, isotopologues of lysine that are spaced 36 mDa apart. Proteins having such close spacing cannot be distinguished during a medium resolution scan, but upon acquiring a high-resolution scan, the two forms of the protein with each amino acid are resolved and the quantitative information revealed. An additional benefit NeuCode SILAC provides for top down is that the spacing of the isotope peaks indicates the number of lysines present in the protein, information that aids in identification. We used NeuCode SILAC to quantify several hundred isotope distributions, manually identify and quantify proteins from 1:1, 3:1, and 5:1 mixed ratios, and demonstrate MS2-based quantitation using ETD.
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