Quantitative Analysis Of Protein Carboxyl Phosphorylation Using Stable Isotope Labeled Amino Phosphonates And Mass Spectrometry

Protein phosphorylation is one of the most important post-translational modifications in living cells.It participates in cell cycle control,receptor-mediated signal transduction,differentiation,proliferation,transformation,and metabolism.Thousands of phosphorylation sites have been identified in different types of cells and organisms through antibodies specific to phosphorylation and proteomics.The research mainly focuses on the phophorylation of serine,threonine and tyrosine in eukaryotes.However,the carboxyl-phosphorylated forms of phosphorylated aspartic acid and phosphorylated glutamate are difficult to be widely discovered and dynamically quantified due to their chemical and thermal instability.In this paper,the labeling reagent AEP and its stable isotope labeling reagent 4D-AEP are designed and synthesized,which can quickly capture the electrophilic acyl phosphate groups specific to phosphorylated aspartic acid sites and phosphorylated glutamate sites in the bacterial proteome,for specific recognition and extensive discovery of the carboxyl phosphorylation site in the proteome.The structure of the synthesized labeling reagent was characterized and optimized by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry.The structure contains a nucleophilic amino group and a phosphate ester group with a mass spectrometry sensitization effect.The synthesis of the stable isotope labeling reagent is achieved by introducing deuterium atoms into the phosphate ester group.Two stable modifications can be generated at the carboxyl phosphorylation site:AEP(+163.0938 Da)and 4D-AEP(+167.1189 Da).According to the addition of stable modifications on the peptide,the labeled peptide and phosphorylation site can be quickly identified.The response regulator protein KDPE and VICR of Escherichia coli were used as experimental objects,firstly,the in vitro phosphorylated pKdpE and pVicR were obtained by Acetyl phosphate(AcP)treatment.Then the protein labeling conditions were optimized,and it was found that the best labeling effect could be obtained at room temperature.Under this labeling condition,the AEP modification was successfully added to the known pAsp52 sites of the pKdpE and pVicR,and through the identification verification with the LC-MS/MS detection and proteomics analysis,established a fast proteomics method to recognize and identify the phosphorylated aspartic acid sites in proteins.This approach can also be applied to the discovery of phosphorylated glutamate sites.Further dynamic quantification of carboxyl-phosphorylated proteins can be achieved by introducing a stable isotope labeled reagent 4D-AEP.In summary,this study provides a new method for specific identification and extensive discovery of unstable carboxylic phosphorylation modifications and for revealing the biological functions of carboxylic phosphorylated proteins.