Phosphorylation is one of the most common posttranslational modification (PTM) of proteins. Main challenge of phosphoprotein detection is their low abundance comparing to abundance of unmodified proteins. The method of selected reactions monitoring (SRM) allows to perform very sensitive and selective analysis of desired PTMs. Using myelin basic protein (MBP) as a model we have developed a method for phosphoprotein detection by SRM. The method is based on obtaining of phosphoproteins in a reconstituted kinase system and following usage these phosphorylated protein as a template for the development of the SRM method. The developed method was successfully applied for detection of phosphopeptides of myelin basic protein in the samples of human brain glioma.
Fosforilirovanie iavliaetsia samoĭ rasprostranennoĭ i naibolee znachimoĭ post-transliatsionnoĭ modifikatsieĭ (PTM) belkov. Issledovanie fosfoform belkov znachitel'no zatrudneno iz-za ikh nizkoĭ kontsentratsii v biologicheskikh obraztsakh v sravnenii s nativnymi belkami. Primenenie metodov monitoringa vybrannykh reaktsiĭ (SRM) pozvoliaet provodit' napravlennyĭ analiz PTM s vysokoĭ spetsifichnost'iu i chuvstvitel'nost'iu. Na primere osnovnogo belka mielina (MBP) prodemonstrirovan metodicheskiĭ podkhod, osnovannyĭ na poluchenii fosforilirovannogo belka v rekonstruirovannoĭ kinaznoĭ sisteme i dal'neĭshem ispol'zovanii ego tripticheskikh fosfopeptidov v kachestve standartov dlia SRM metoda. Razrabotannyĭ metod byl uspeshno primenen dlia opredeleniia saĭtov fosforilirovaniia v tkani gliomy mozga cheloveka.
Keywords: post-translational modifications (PTM); protein phosphorylation; proteomics; selected reaction monitoring (SRM).