title: Quantitative proteomics using reductive dimethylation for stable isotope labeling.
abstract: Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.
authors.affiliation: CEA, DSV, IG, Genoscope; CNRS-UMR8030, Évry, France; Université d'Évry Val d'Essonne; atolonen@genoscope.cns.fr.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Tolonen
authors.affiliation: Massachusetts General Hospital Cancer Center; whaas@mgh.harvard.edu.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Haas
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D003013:Clostridium / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D008745:Methylation; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D005557:Formaldehyde; D003903:Deuterium