title: The impact of carbon-13 and deuterium on relative quantification of proteins using stable isotope diethyl labeling.
abstract: BACKGROUND
  Stable isotopic labeling techniques are useful for quantitative proteomics. A cost-effective and convenient method for diethylation by reductive amination was established. The impact using either carbon-13 or deuterium on quantification accuracy and precision was investigated using diethylation.
  
  
  METHODS
  We established an effective approach for stable isotope labeling by diethylation of amino groups of peptides. The approach was validated using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nanospray liquid chromatography/electrospray ionization (nanoLC/ESI)-ion trap/orbitrap for mass spectrometric analysis as well as MaxQuant for quantitative data analysis.
  
  
  RESULTS
  Reaction conditions with low reagent costs, high yields and minor side reactions were established for diethylation. Furthermore, we showed that diethylation can be applied to up to sixplex labeling. For duplex experiments, we compared diethylation in the analysis of the proteome of HeLa cells using acetaldehyde-(13) C(2)/(12) C(2) and acetaldehyde-(2) H(4)/(1) H(4). Equal numbers of proteins could be identified and quantified; however, (13) C(4)/(12) C(4) -diethylation revealed a lower variance of quantitative peptide ratios within proteins resulting in a higher precision of quantified proteins and less falsely regulated proteins. The results were compared with dimethylation showing minor effects because of the lower number of deuteriums.
  
  
  CONCLUSIONS
  The described approach for diethylation of primary amines is a cost-effective and accurate method for up to sixplex relative quantification of proteomes. (13) C(4)/(12) C(4) -diethylation enables duplex quantification based on chemical labeling without using deuterium which reduces identification of false-negatives and increases the quality of the quantification results.
authors.affiliation: Biotechnology Centre of Oslo, University of Oslo, P. O. Box 1125 Blindern, 0317, Oslo, Norway.|Department of Biosciences, University of Oslo, P. O. Box 1066 Blindern, 0316, Oslo, Norway.
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Koehler
authors.affiliation: Biotechnology Centre of Oslo, University of Oslo, P. O. Box 1125 Blindern, 0317, Oslo, Norway.|Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, 1432, Ås, Norway.
authors.forename: Magnus Ø
authors.initials: MØ
authors.lastname: Arntzen
authors.affiliation: Biotechnology Centre of Oslo, University of Oslo, P. O. Box 1125 Blindern, 0317, Oslo, Norway.|Department of Biosciences, University of Oslo, P. O. Box 1066 Blindern, 0316, Oslo, Norway.
authors.forename: Bernd
authors.initials: B
authors.lastname: Thiede
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D002247:Carbon Isotopes; D020543:Proteome; D003903:Deuterium