Details zur Publikation

Kategorie Textpublikation
Referenztyp Zeitschriften
DOI 10.1002/rcm.3290
Titel (primär) Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell culture (SILAC)
Autor Schmidt, F.; Strozynski, M.; Salus, S.S.; Nilsen, H.; Thiede, B.
Quelle Rapid Communications in Mass Spectrometry
Erscheinungsjahr 2007
Department PROTEOM
Band/Volume 21
Heft 23
Seite von 3919
Seite bis 3926
Sprache englisch
Abstract 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 36 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 (c) 2007 John Wiley & Sons, Ltd
dauerhafte UFZ-Verlinkung
Schmidt, F., Strozynski, M., Salus, S.S., Nilsen, H., Thiede, B. (2007):
Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell culture (SILAC)
Rapid Commun. Mass Spectrom. 21 (23), 3919 - 3926 10.1002/rcm.3290