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Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics

Abstract

Accurate quantification of protein expression in biological systems is an increasingly important part of proteomics research. Incorporation of differential stable isotopes in samples for relative protein quantification has been widely used. Stable isotope incorporation at the peptide level using dimethyl labeling is a reliable, cost-effective and undemanding procedure that can be easily automated and applied in high-throughput proteomics experiments. Although alternative multiplex quantitative proteomics approaches introduce isotope labels at the organism level ('stable isotope labeling by amino acids in cell culture' (SILAC)) or enable the simultaneous analysis of eight samples (isobaric tagging for relative and absolute quantification (iTRAQ)), stable isotope dimethyl labeling is advantageous in that it uses inexpensive reagents and is applicable to virtually any sample. We describe in-solution, online and on-column protocols for stable isotope dimethyl labeling of sample amounts ranging from sub-micrograms to milligrams. The labeling steps take approximately 60–90 min, whereas the full protocol including digestion and (two-dimensional) liquid chromatography-mass spectrometry takes approximately 1.5–3 days to complete.

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Figure 1: Labeling schemes of triplex stable isotope dimethyl labeling.
Figure 2: Pictures of various experimental set-ups for stable isotope dimethyl labeling.
Figure 3: Extracted ion chromatograms and mass spectra of the BSA peptide YICDNQDTISSK.
Figure 4: MS triplets of doubly and triply charged triplex stable isotope dimethyl-labeled BSA peptides LGEYGFQNALIVR and TCVADESHAGCEK.
Figure 5: Differential protein expression and phosphorylation after morpholino-mediated Fyn/Yes knockdown in zebrafish embryos.

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Acknowledgements

We thank Ms Sharon Gauci for critically reviewing this paper. This study was supported by The Netherlands Proteomics Centre (http://www.netherlandsproteomicscentre.nl/).

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Correspondence to Shabaz Mohammed or Albert J R Heck.

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Boersema, P., Raijmakers, R., Lemeer, S. et al. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4, 484–494 (2009). https://doi.org/10.1038/nprot.2009.21

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