HL-60 promyelocytic cells are a widely used as a model for studying induced granulocytic differentiation. Investigation of proteins of the nuclear fraction, particularly transcription factors, is necessary for a better understanding of molecular mechanisms of cell maturation. Mass spectrometry is a powerful tool for analyzing a proteome due to its high sensitivity, specificity and performance. In this paper, using the selected reaction monitoring (SRM) method, we have assessed the levels of RBPJ, STAT1, CEBPB, CASP3, VAV1, PRKDC, PARP1 and UBC9 nuclear proteins isolated using hypertonic buffer, detergents (sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and fissionable detergent ProteaseMAX™) and using centrifugation in a sucrose density gradient. The minimum and maximum protein content was 1.13±0.28 and 14.34±1.63 fmol/mkg of total protein for the transcription factor RBPJ and ubiquitin-protein ligase type I UBC9, respectively. According to the results of shotgun mass spectrometric analysis of nuclear fractions, 2356 proteins were identified, of which 106 proteins were annotated as transcription factors. 37 transcription factors were uniquely identified in the fraction obtained by centrifugation in a sucrose density gradient, while only 9 and 8 transcription factors were uniquely identified in the nuclear fractions obtained using hypertonic buffer and detergents, respectively. The transcription factors identified in the HL-60 cell line represent regulatory molecules; their directed profiling under the influence of differentiation inducers, will shed light on the mechanism of granulocyte maturation.
Promielotsitarnye kletki linii HL-60 iavliaiutsia shiroko ispol'zuemoĭ model'iu dlia izucheniia indutsiruemoĭ granulotsitarnoĭ differentsirovki. Issledovanie belkov iadernoĭ fraktsii, v osobennosti transkriptsionnykh faktorov, neobkhodimo dlia luchshego ponimaniia molekuliarnykh mekhanizmov sozrevaniia kletok. Mass-spektrometriia iavliaetsia moshchnym metodom dlia analiza proteoma v silu vysokoĭ chuvstvitel'nosti, proizvoditel'nosti i spetsifichnosti analiza. V rabote, s ispol'zovaniem metoda monitoringa vybrannykh reaktsiĭ (SRM), provedena otsenka urovnia soderzhaniia iadernykh belkov RBPJ, STAT1, CEBPB, CASP3, VAV1, PRKDC, PARP1 i UBC9, vydelennykh s primeneniem gipertonicheskogo bufera, detergentov (dodetsilsul'fata natriia (SDS), dezoksikholata natriia (DOC) i rasshchepliaemogo detergenta ProteaseMAX™) i s pomoshch'iu tsentrifugirovaniia v gradiente plotnosti sakharozy. Minimal'noe i maksimal'noe soderzhanie belkov sostavilo 1,13±0,28 i 14,34±1,63 fmol'/mkg obshchego belka dlia transkriptsionnogo faktora RBPJ i ubikvitin-proteinligazy pervogo tipa UBC9 sootvetstvenno. Po rezul'tatam panoramnogo mass-spektrometricheskogo analiza iadernykh fraktsiĭ udalos' identifitsirovat' 2356 belkov, iz nikh 106 belkov byli annotirovany kak transkriptsionnye faktory. Tret' transkriptsionnykh faktorov (37 belkov) byli identifitsirovany tol'ko vo fraktsii, poluchennoĭ s pomoshch'iu tsentrifugirovaniia v gradiente plotnosti sakharozy, v to vremia kak lish' 9 i 8 transkriptsionnykh faktorov bylo identifitsirovano tol'ko vo fraktsiiakh iader, poluchennykh s ispol'zovaniem gipertonicheskogo bufera i detergentov, sootvetstvenno. Transkriptsionnye faktory, identifitsirovannye v kletkakh linii HL-60, predstavliaiut soboĭ reguliatornye molekuly, napravlennoe profilirovanie kotorykh pod deĭstviem induktorov differentsirovki pozvolit prolit' svet na mekhanizm sozrevaniia granulotsitov.
Keywords: HL-60 cell line; nuclear proteome; selected reaction monitoring (SRM); shotgun mass spectrometry; transcription factors.