GingisREX® in a Bottom-Up Analysis of Histones

Bottom-up mass spectrometry where the intact protein is digested into smaller peptides is an indispensable tool for proteomics and the studying of histone codes. Since histones are enriched with the basic amino acids arginine and lysine, enzymatic digestion is often challenging as peptides too short for LC retention and PTM localization are generated. In addition, the complex workflow entails that the combination of samples preparation, data acquisition and analysis provides a different image of the histone code.

In this paper, Daled and colleagues at Ghent University and Max Planck Institute present a new perspective on histone sample preparation and provide a roadmap that guides the selection of a protocol to cover certain areas of interest. Using bovine histone standards, the scientists evaluated four different sample preparation workflows, including trypsin digests with and without derivatization, ArgC digestion, and a workflow with the arginine specific GingisREX (RgpB) enzyme.

The results showed that the high specificity of both trypsin and GingisREX clearly displayed the benefits of these enzymes for both histone and proteomics workflows, in contrast to the less specific ArgC enzyme that hampered accurate peptide and proteoforms quantification. The authors conclude that while trypsin digestion largely remains preferred in sample preparation, GingisREX is a complementary tool that provides the benefit of retaining all biological histone PTMs while minimizing the chemical noise otherwise attributed to propionylation.

Reference

Daled et al., 2021. Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions. Proteomes. https://doi.org/10.1038/s41598-021-90966-1.

GingisREX – Arginie-specific protein digestion