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Free keywords:
MIDDLE-DOWN PROTEOMICS; QUANTITATIVE ASSESSMENT; CYSTEINE PROTEINASE;
SIDE REACTIONS; PROPIONYLATION; PERFORMANCE; DERIVATIZATION;
PURIFICATION; GINGIPAIN; CODEBiochemistry & Molecular Biology; histone code; coverage; epigenetics; mass spectrometry; sample
preparation; workflow optimization; GingisREX;
Abstract:
Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment between runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX(R), Genovis, Lund, Sweden), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues. Raw data are available via ProteomeXchange with identifier PXD024423.
