Chemical Labeling Method Based on Amidation for the Analysis of Phosphopeptides

Post-translational modification of proteins is complex and diverse, with the phosphorylation regulated by kinases and phosphatases being one of the most widespread and important modifications. Dynamic change of quantitative analysis of phosphorylated proteins is helpful to clarify the mechanism of phosphorylation regulating life activities and understand its role in achieving biological functions. Mass spectrometry is the most commonly used strategy for the quantitative analysis of post-translational modification, involving label-free quantification, internal standard absolute quantification, and chemical labeling, etc. In this paper, a chemical labeling approach which has been used for protein glycosylation profiling was applied to the relative quantitative analysis of phosphopeptides. Using the method, carboxyl groups were located on the side chain of aspartic acid and glutamic acid residues, and the C-terminus of peptide were allowed to react with dimethylamine (d0-DMA)/deuterated dimethylamine (d6-DMA). As a result, the carboxyl groups were neutralized, and all enzymatic peptides were labeled with light/heavy tags. After the samples with different tags were mixed, the phosphopeptides were enriched using TiO₂ magnetic particles and then analyzed qualitatively and relatively quantitatively by liquid chromatography-mass spectrometry (LC-MS). Amidation could shield the negatively charged carboxyl groups of non-phosphorylated and phosphorylated peptides, so that the peptides with phosphate groups mainly exhibited negative charge property, thus improving the enrichment specificity of TiO₂ for phosphopeptides as well as its detection sensitivity in LC-MS analysis. Using 0.2 mg casein as a standard, 8 phosphopeptides were detected in the derivatized samples. A high abundance of b and y ions indicated that the amidation by dimethylamine was highly specific and usually occurs only at the carboxyl group without a significant effect on the phosphate group of peptides. A series of molar ratios (5∶1, 2∶1, 1∶1, 1∶2, 1∶5) of samples were derivatized using d0-DMA and d6-DMA, respectively. The results showed that the error was less than 20% for each ratio, indicating that the method possessed good relative quantitative range and accuracy. However, too many derivative sites in the peptides could result in incomplete derivatization, and the C-terminal lysine residues might self-condense to form lactam during the derivatization process. By selecting some individualized proteases during protein digestion, such as GluC, the number of derivative sites might be in an appropriate range and lysine residue could be avoided at the terminal of peptides, which should be useful for improving identification efficiency and quantitative accuracy of phosphopeptides. In summary, the chemical labeling method based on dimethylamine derivatization presents advantages for high-throughput relative quantitative analysis of phosphopeptides and it is expected to be a powerful tool for protein phosphorylation profiling in biological tissues.