Novikova S E, Vakhrushev I V, Tsvetkova A V, Shushkova N A, Farafonova T E, Yarygin K N, Zgoda V G
Institute of Biomedical Chemistry, Moscow, Russia.
Biomed Khim. 2019 Jun;65(4):294-305. doi: 10.18097/PBMC20196504294.
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.
HL-60早幼粒细胞被广泛用作研究诱导粒细胞分化的模型。研究核组分中的蛋白质，尤其是转录因子，对于更好地理解细胞成熟的分子机制至关重要。由于具有高灵敏度、特异性和性能，质谱分析法是分析蛋白质组的强大工具。在本文中，我们使用选择反应监测（SRM）方法，评估了使用高渗缓冲液、去污剂（十二烷基硫酸钠（SDS）、脱氧胆酸钠（DOC）和可裂解去污剂ProteaseMAX™）并通过蔗糖密度梯度离心分离得到的核蛋白RBPJ、STAT1、CEBPB、CASP3、VAV1、PRKDC、PARP1和UBC9的水平。转录因子RBPJ和I型泛素蛋白连接酶UBC9的最低和最高蛋白质含量分别为1.13±0.28和14.34±1.63 fmol/mkg总蛋白。根据对核组分的鸟枪法质谱分析结果，共鉴定出2356种蛋白质，其中106种蛋白质被注释为转录因子。在通过蔗糖密度梯度离心获得的组分中唯一鉴定出37种转录因子，而在使用高渗缓冲液和去污剂获得的核组分中分别仅唯一鉴定出9种和8种转录因子。在HL-60细胞系中鉴定出的转录因子代表调控分子；在分化诱导剂的影响下对它们进行定向分析，将有助于阐明粒细胞成熟的机制。
