Absolute quantification of transcription factors in human erythropoiesis using selected reaction monitoring mass spectrometry

• Published in: STAR protocols 2020-12, Vol.1 (3), p.100216-100216, Article 100216
• Main Authors: Gillespie, Mark A., Palii, Carmen G., Sanchez-Taltavull, Daniel, Perkins, Theodore J., Brand, Marjorie, Ranish, Jeffrey A.
• Format: Article
• Language: English
• Subjects: Cell Differentiation; Cell Differentiation - genetics; Erythropoiesis - physiology; Gene Expression Regulation - genetics; Gene Regulatory Networks - genetics; Hematopoietic Stem Cells - metabolism; Humans; Mass Spectrometry; Mass Spectrometry - methods; Proteomics; Proteomics - methods; Protocol; Transcription Factors - analysis
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2020.100216

Quantitative changes in transcription factor (TF) abundance regulate dynamic cellular processes, including cell fate decisions. Protein copy number provides information about the relative stoichiometry of TFs that can be used to determine how quantitative changes in TF abundance influence gene regulatory networks. In this protocol, we describe a targeted selected reaction monitoring (SRM)-based mass-spectrometry method to systematically measure the absolute protein concentration of nuclear TFs as human hematopoietic stem and progenitor cells differentiate along the erythropoietic lineage. For complete details on the use and execution of this protocol, please refer to Gillespie et al. (2020). [Display omitted] •Protocol for absolute quantification of TFs in human erythropoiesis•Selected reaction monitoring mass-spectrometry parameters for each peptide•Validated SRM assays corresponding to >100 TFs•Copy number reveals the relative stoichiometries of TFs during erythropoiesis Quantitative changes in transcription factor (TF) abundance regulate dynamic cellular processes, including cell fate decisions. Protein copy number provides information about the relative stoichiometry of TFs that can be used to determine how quantitative changes in TF abundance influence gene regulatory networks. In this protocol, we describe a targeted selected reaction monitoring (SRM)-based mass spectrometry method to systematically measure the absolute protein concentration of nuclear TFs as human hematopoietic stem and progenitor cells differentiate along the erythropoietic lineage.