title: Testing Suitability of Cell Cultures for SILAC-Experiments Using SWATH-Mass Spectrometry.
abstract: Precise quantification is a major issue in contemporary proteomics. Both stable-isotope-labeling and label-free methods have been established for differential protein quantification and both approaches have different advantages and disadvantages. The present protocol uses the superior precision of label-free SWATH-mass spectrometry to test for suitability of cell lines for a SILAC-labeling approach as systematic regulations may be introduced upon incorporation of the "heavy" amino acids. The SILAC-labeled cell cultures can afterwards be used for further analyses where stable-isotope-labeling is mandatory or has substantial advantages over label-free approaches such as pulse-chase-experiments and differential protein interaction analyses based on co-immunoprecipitation. As SWATH-mass spectrometry avoids the missing-value-problem typically caused by undersampling in highly complex samples and shows superior precision for the quantification, it is better suited for the detection of systematic changes caused by the SILAC-labeling and thus, can serve as a useful tool to test cell lines for changes upon SILAC-labeling.
authors.affiliation: Department of Biochemistry I, University of Regensburg, Regensburg, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Reinders
authors.affiliation: Institute of Pathology, University of Regensburg, Regensburg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Völler
authors.affiliation: Institute of Pathology, University of Regensburg, Regensburg, Germany.|Institute of Biochemistry, Emil-Fischer-Zentrum, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Anja-K
authors.initials: AK
authors.lastname: Bosserhoff
authors.affiliation: Institute of Functional Genomics, University of Regensburg, Josef-Engert-Str. 9, 93053, Regensburg, Germany.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Oefner
authors.affiliation: Institute of Functional Genomics, University of Regensburg, Josef-Engert-Str. 9, 93053, Regensburg, Germany. joerg.reinders@klinik.uni-regensburg.de.
authors.forename: Jörg
authors.initials: J
authors.lastname: Reinders
mesh_terms: D000596:Amino Acids; D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D020543:Proteome
keywords: Label-free proteomics; Melanoma cells; SILAC; SWATH-MS