Identification Of The Substrates Of Arginine Kinase McsB Using ?-[¹⁸O₄]-ATP And Isoleucine Assisted Differentiation Of Disaccharide Isomers By ESI-MS²

The reversible phosphorylation of proteins has been recognized as one of the most important post-translational modifications in organisms since it has been discovered.It exists in many biological processes and participates in many cellular activities such as metabolism,cell growth,cell signal transduction and other important biological processes.Arginine phosphorylation is a phosphoramidate modification in the form of P-N bond that occurs on the side chain of the arginine.However,since the high energy and acid labile nature of P-N bond,the research on arginine phosphorylation does not have breakthrough.In recent years,with the discovery of the first arginine kinase McsB and the development of proteomics,the study on arginine phosphorylation has been paid more and more attention.Systematic identification of McsB substrates can not only help understand the biological function of phosphoarginine proteins,but also give an insight in understanding the biochemical processes arginine kinase McsB involved in.In this paper,a novel ?-[18O4]-ATP stable isotope labeling method was developed for the systematic identification of McsB substrates.We selected the total protein of Geobacillus stearothermophilus as the substrates library,then we applied in vitro kinase reaction using McsB with high enzyme activity to phosphorylate its substrates library for the first time.After trypsin digestion and mild TiO2 enrichment we finally identified the substrates of McsB by Nano LC-MS.The labeling of the McsB substrate proteins by ?-[18O4]-ATP cause a 6 Da mass shift of the labeled proteins compared with other phosphorylated proteins,thereby this can be used to effectively distinguish the substrates of kinase McsB from other phosphorylated proteins.With repeated experiments,we now identified 21 phosphoarginine sites containing in 20 phosphoarginine proteins.At present,this part of work is still optimized for identifying more phosphoarginine proteins.Therefore,a novel method for the identification of arginine kinase substrates using stable isotope labeling was established.The method can also be widely used in the identification of other kinase substrates.Carbohydrate is an important source of energy for life,and is also involved in a series of biological processes.Glycosylation of proteins is another form of post-translational modification that plays a very important role in the process of cell-to-cell interactions and signal transduction.At the same time,saccharides can also form sugar conjugates with proteins,lipids,etc.These ligands can anchor the cells and also function in identifying physiological pathologies.The identification of disaccharides has always been a challenge since there are many disaccharides isomers.When we study the interactions between isoleucine and disaccharides,we found that[Ile+Dis+K]+ complex was formed.Through mass spectrometry based analysis we found that the non-covalent complex could provide sugar skeleton structural information.Finally,the disaccharide isomers were successfully identified based on the differences between the ion fragments and the peak intensity of the eight different disaccharide isomers complex with Ile and potassium ion by ESI-MS2 combined with principle compenents analysis.