Abstract
(1) Background: The proteomic analysis of histones constitutes a delicate task due to the combination of two factors: slight variations in the amino acid sequences of variants and the multiplicity of post-translational modifications (PTMs), particularly those occurring on lysine residues. (2) Methods: To dissect the relationship between both aspects, we carefully evaluated PTM identification on lysine 27 from histone H3 (H3K27) and the artefactual chemical modifications that may lead to erroneous PTM determination. H3K27 is a particularly interesting example because it can bear a range of PTMs and it sits nearby residues 29 and 31 that vary between H3 sequence variants. We discuss how the retention times, neutral losses and immonium/diagnostic ions observed in the MS/MS spectra of peptides bearing modified lysines detectable in the low-mass region might help validate the identification of modified sequences. (3) Results: Diagnostic ions carry key information, thereby avoiding potential mis-identifications due to either isobaric PTM combinations or isobaric amino acid-PTM combinations. This also includes cases where chemical formylation or acetylation of peptide N-termini artefactually occurs during sample processing or simply in the timeframe of LC-MS/MS analysis. Finally, in the very subtle case of positional isomers possibly corresponding to a given mass of lysine modification, the immonium and diagnostic ions may allow the identification of the in vivo structure.
Highlights
Within a living organism, one cell type is differentiated from another by its own gene expression program
A wealth of post-translational modifications (PTMs) have been described over the past decade to possibly modify histone lysines, which renders the task of correctly identifying the combination of peptide sequence and PTMs very challenging
We wanted to assess whether some analytical features could help ascertain the identity of modified histone peptides
Summary
One cell type is differentiated from another by its own gene expression program. The previous differences are compensated by the change of amino acids in the three above variants, making the sequences H3mm13-Kac SVPSTGGVK, H3.3-Kbut SAPSTGGVK and H3canoKohbu SAPATGGVK strictly isobaric because they have the same elemental composition, and cannot be distinguished even by latest-generation MS instruments offering mass accuracies below 1 ppm. Beside these subtle amino acid variations, chemical artefacts can contribute to the mis-identification of modified histone peptides. We carried out this study using histones extracted from mouse testis, because numerous variants are expressed in this organ which are decorated with a wealth of PTMs being dynamically regulated during spermatogenesis [20]
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