Proteomics of Transcription Factors: Identification of a Pool of HL-60 Cell Line-Specific Regulatory Proteins
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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 proteomes due to its high sensitivity, specificity and performance. In this study performed 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 cleavable 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/μg 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 treatment of differentiation inducers, will shed light on the mechanism of granulocyte maturation.
Keywords: nuclear proteome selected reaction monitoring (SRM) shotgun mass spectrometry transcription factors HL-60 cell line
Experiments were performed in the Center for Collective Use “Human Proteome” (Institute of Biomedical Chemistry, Moscow).
The study was financially supported by the Russian Science Foundation (project no. 17-75-10201).
COMPLIANCE WITH ETHICAL STANDARDS
This article does not contain any research involving humans or using animals as objects.
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