Stable isotope labeling-based two-step derivatization strategy for analysis of Phosphopeptides

Investigating site-specific protein phosphorylation remains a challenging task. The present study introduces a two-step chemical derivatization method for accurate identification of phosphopeptides. Methylamine neutralizes carboxyl groups, thus reducing the adsorption of non-phosphorylated peptides during enrichment, while dimethylamine offers a cost-effective reagent for stable isotope labeling of phosphorylation sites. The derivatization improves the mass spectra obtained through liquid chromatography-tandem mass spectrometry. The product ions at m/z 58.07 and 64.10 Da, resulting from dimethylamine-d₀ and dimethylamine-d₆ labeled phosphorylation sites respectively, can serve as report ions. Derivatized phosphopeptides from casein demonstrate enhanced ionization and formation of product ions, yielding a significant increase in the number of identifiable peptides. When using the parallel reaction monitoring technique, it is possible to distinguish isomeric phosphopeptides with the same amino acid sequence but different phosphorylation sites. By employing a proteomic software and screening the report ions, we identified 29 endogenous phosphopeptides in 10 μL of human saliva with high reliability. These findings indicate that the two-step derivatization strategy has great potential in site-specific phosphorylation and large-scale phosphoproteomics research. SIGNIFICANCE: There is a significant need to improve the accuracy of identifying phosphoproteins and phosphopeptides and analyzing them quantitatively. Several chemical derivatization techniques have been developed to label phosphorylation sites, thus enabling the identification and relative quantification of phosphopeptides. Nevertheless, these methods have limitations, such as incomplete conversion or the need for costly isotopic reagents. Building upon previous contributions, our study moves the field forward due to high efficiency in site-specific labeling, cost-effectiveness, improved sensitivity, and comprehensive product ion coverage. Using the two-step derivatization approach, we successfully identified 29 endogenous phosphopeptides in 10 μL of human saliva with high reliability. The outcomes underscore the possibility of the method for site-specific phosphorylation and large-scale phosphoproteomics investigations.