Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates

Abstract

Stable isotope labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope labeling has been performed at both the protein as well as the peptide level using either metabolic or chemical labeling. Here, we present a straightforward and cost-effective triplex quantification method that is based on stable isotope dimethyl labeling at the peptide level. Herein, all proteolytic peptides are chemically labeled at their alpha- and epsilon-amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical "shotgun" proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 microg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.

Use of Stable Isotope Dimethyl Labeling Coupled to Selected Reaction Monitoring to Enhance Throughput by Multiplexing Relative Quantitation of Targeted Proteins

“…Since three labeling reactions contribute to the monitored unlabeled portion, our results are comparable to other reports of dimethyl labeling efficiency of 97% or better. 21,[27][28][29] This suggests that incomplete dimethyl labeling of peptides is not a critical issue impeding SRM assays. Table S-4A.…”

In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling

“…For intermediate labels, formaldehyde-D 2 (573 mol) was used. For the heavy labeling, 13 C-D 2 -formaldehyde (573 mol) and freshly prepared cyanoborodeuteride (278 mol) were used (20,21). The light, intermediate, and heavy dimethyl-labeled samples were mixed in 1:1:1 ratio based on total peptide amount, which was determined by running an aliquot of the labeled samples on a regular LC-MS run and comparing overall peptide signal intensities.…”

“…Quantification-Quantification of peptide triplets of which at least one had a Mascot peptide score of 20 was performed using an in-house dimethyl-adapted version of MSQuant (SourceForge) as described previously (20). Briefly, peptide ratios were obtained by calculating the extracted ion chromatograms (XICs) of the "light," "intermediate," and "heavy" forms of the peptide using the monoisotopic peaks only.…”

SILAC for biomarker discovery

“…First, 15 N-substituted media is difficult to make for mammalian cells that require more complex components. Second, since the mass shift introduced by SIL depends on the number of isotopically-altered elements in the analyte, it is typically different 5 for different peptides, greatly complicating data analysis. Finally, due to the incorporation of multiple labels per analyte, even very highly enriched isotopes result in partial labeling.…”

“…Thus, mass spectrometry-based proteomics is intrinsically not quantitative. However, over the last decade several strategies have been developed that enable quantification on a global proteomic scale [2][3][4][5]. The general principle of these strategies is that relative changes in the intensity of a peak rather than its absolute intensity are used for quantification.…”