December 13, 2002Journal of Proteome Research
Properties of 13C-Substituted Arginine in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)
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Discussion
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Implication
Experimental study demonstrates high-accuracy quantitative proteomics using 13C-arginine SILAC in cell culture lysates, indicating improved mass spectrometry reproducibility.
Key Points
- To evaluate the performance, chromatographic behavior, and quantitation accuracy of fully substituted 13C-labeled arginine in stable isotope labeling by amino acids in cell culture (SILAC).
- Metabolically labeled cell cultures with fully substituted 13C-arginine followed by tryptic digestion to yield C-terminal labeled peptides.
- Resolved mixtures of total cell lysates mixed in known ratios using one-dimensional SDS-PAGE.
- Analyzed peptide mixtures via liquid chromatography-tandem mass spectrometry (LC-MS/MS) to monitor coelution and fragment ion shifts.
- Labeled and unlabeled peptides coeluted during liquid chromatography, avoiding quantitation error caused by retention-time shifts.
- Tandem mass spectra showed a predictable mass shift on C-terminal y-ions, aiding database identification.
- Quantitation of cell lysate mixtures yielded reproducible measurements, achieving relative standard deviations better than 5% under optimal conditions.
Cite This Study
Ong et al. (2002) studied this question.