title: Effective correction of experimental errors in quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC).
abstract: Accurate and reliable quantitative proteomics in cell culture has been considerably facilitated by the introduction of the stable isotope labeling by amino acids in cell culture (SILAC), combined with high resolution mass spectrometry. There are however several major sources of quantification errors that commonly occur with SILAC techniques, i.e. incomplete incorporation of isotopic amino acids, arginine-to-proline conversion, and experimental errors in final sample mixing. Dataset normalization is a widely adopted solution to such errors, however this may not completely prevent introducing incorrect expression ratios. Here we demonstrate that a label-swap replication of SILAC experiments was able to effectively correct experimental errors by averaging ratios measured in individual replicates using quantitative proteomics and phosphoproteomics of ligand treatment of neural cell cultures. Furthermore, this strategy was successfully applied to a SILAC triplet experiment, which presents a much more complicated experimental matrix, affected by both incomplete labeling and arginine-to-proline conversion. Based on our results, we suggest that SILAC experiments should be designed to incorporate label-swap replications for enhanced reliability in expression ratios.
authors.affiliation: Receptor Pharmacology Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD, United States.
authors.forename: Sung-Soo
authors.initials: SS
authors.lastname: Park
authors.forename: Wells W
authors.initials: WW
authors.lastname: Wu
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.forename: Rong-Fong
authors.initials: RF
authors.lastname: Shen
authors.forename: Bronwen
authors.initials: B
authors.lastname: Martin
authors.forename: Stuart
authors.initials: S
authors.lastname: Maudsley
mesh_terms: D000465:Algorithms; D016477:Artifacts; D018929:Cell Culture Techniques / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D020543:Proteome