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Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell culture (SILAC)
Author(s) -
Schmidt Frank,
Strozynski Margarita,
Salus Sandra S.,
Nilsen Hilde,
Thiede Bernd
Publication year - 2007
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3290
Subject(s) - stable isotope labeling by amino acids in cell culture , chemistry , amino acid , peptide , hela , lysine , quantitative proteomics , arginine , isobaric labeling , tandem mass spectrometry , mass spectrometry , biochemistry , trifluoroacetic acid , tandem mass tag , chromatography , proteomics , protein mass spectrometry , cell , gene
Stable isotope labeling with amino acids in cell culture (SILAC) has evolved to be a major technique for quantitative proteomics using cell cultures. We developed a rapid method to follow and determine the incorporation of arginine and lysine. Analysis of the heavy state is required to avoid quantification errors. Moreover, the mixture of light and heavy states can be exploited to normalize the protein amount for subsequent relative quantification experiments. Therefore, peptides from different cell lines were extracted with 0.1% trifluoroacetic acid and analyzed by matrix‐assisted laser desorption/ionization tandem time‐of‐flight (MALDI‐TOF/TOF) mass spectrometry (MS). This analysis was highly reproducible and was performed in less than 2 h, significantly faster than other methods for the same purpose. Similar peptide mass profiles were obtained for human EBV‐transformed B, Jurkat T, and HeLa cells as well as for mouse embryonic fibroblasts. Proteolytic fragments of 27 human proteins were identified with 56 peptides by MALDI‐MS/MS and can be used as a database for these kinds of experiments. Sequencing revealed that the peptides were predominantly amino‐ and carboxy‐terminal protein fragments displaying a specificity characteristic of the acidic proteases cathepsin D and E. Many of the identified peptides contained arginine and/or lysine, allowing determination of the incorporation rate of these amino acids. Furthermore, the rate of conversion of arginine into proline could be monitored easily. Copyright © 2007 John Wiley & Sons, Ltd.