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.

In-depth research of multidrug resistance related cell surface glycoproteome in gastric cancer

“…The non-glycopeptides from three cell lines were labeled with triplex stable isotope dimethyl labeling approach according to the published protocol [18]. …”

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

“…Subsequently, on-column stable isotope dimethyl labeling was performed as described previously. 27 For the 'Forward' experiment, the peptides from the unstimulated control sample were labeled with light dimethyl labels and those from the 007-AM stimulated sample with intermediate dimethyl labels. Conversely, for the 'Reverse' experiment the 007-AM stimulated sample was labeled with light dimethyl labels and the unstimulated control sample with intermediate dimethyl labels.…”

pH‐regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments

“…In another paper they also mention negligible effects of buffer change in the range between pH 3.0 and 8.2, in a systematic investigation with formaldehyde [26]. Boersema et al in their recent protocol for triplex experiments with formaldehydes isotopomers, recommend to work in the pH range between 5 and 8.5, while She et al use a pH 8.5 buffer for reductive dimethylation, generating formaldehydes in situ from paraformaldehydes before the addition of the reducing agent [27,28].…”