The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of target protein with aptamers immobilized on a solid phase was studied. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as model target protein. It has been demonstrated that the affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads results in an approximately 10-fold increase of the concentration of target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM is possible without an interference from other peptides present in a tryptic digest.
V rabote issledovano sootnoshenie mezhdu kolichestvom belkovoĭ misheni (targetnogo belka) v slozhnom biologicheskom obraztse i ego kolichestvom, opredeliaemym mass-spektrometricheskim metodom monitoringa vybrannykh reaktsiĭ (MVR) pri affinnom obogashchenii targetnogo belka s ispol'zovaniem aptamerov, immobilizovannykh na tverdoĭ faze. Trombin cheloveka, dobavlennyĭ v izvestnykh kontsentratsiiakh k kletochnym ékstraktam, poluchennym iz bakterial'nykh kletok, byl ispol'zovan kak model'nyĭ targetnyĭ belok. Pokazano, chto affinnoe obogashchenie trombina v kletochnykh ékstraktakh s pomoshch'iu trombin-sviazyvaiushchego aptamera, immobilizovannogo na poverkhnosti magnitnykh mikrosfer, privodit priblizitel'no k 10-kratnomu uvelicheniiu kontsentratsii targetnogo belka i 100-kratnomu snizheniiu nizhnego znacheniia diapazona kontsentratsiĭ, v kotorom vozmozhna ego kolichestvennaia detektsiia metodom MVR bez sushchestvennoĭ interferentsii so storony drugikh peptidov, prisutstvuiushchikh v tripticheskom gidrolizate.
Keywords: aptamers; enrichment; quantitative detection; selected reaction monitoring; thrombin.