Tackling aspecific side reactions during histone propionylation: The promise of reversing overpropionylation

Abstract

Histone proteins are essential elements for DNA packaging. Moreover, the PTMs that are extremely abundant on these proteins, contribute in modeling chromatin structure and recruiting enzymes involved in gene regulation, DNA repair and chromosome condensation. This fundamental aspect, together with the epigenetic inheritance of histone PTMs, underlines the importance of having biochemical techniques for their characterization. Over the past two decades, significant improvements in mass accuracy and resolution of mass spectrometers have made LC-coupled MS the strategy of choice for accurate identification and quantification of protein PTMs. Nevertheless, in previous work we disclosed the limitations and biases of the most widely adopted sample preparation protocols for histone propionylation, required prior to bottom-up MS analysis. In this work, however, we put forward a new specific and efficient propionylation strategy by means of propionic anhydride. In this method, aspecific overpropionylation at serine (S), threonine (T) and tyrosine (Y) is reversed by adding hydroxylamine (HA). We recommend using this method for future analysis of histones through bottom-up MS.

Keywords: Histone; Mass spectrometry; Method optimization; Propionylation; Technology.

Figure 1

Lowering the molar excess (42x, 20x and 5x) of propionic anhydride per primary amine prevents most overpropionylation but increases underpropionylation. Each method is represented by a radar chart showing the average contribution of over‐ (blue), under‐ (yellow) and desired propionylation for seven peptides monitored. Each peptide is located on one angle of the radar chart and each peptide form is represented by another color. The abundance of a specific peptide form is shown on the radius, whereby a conversion rate of 0 is located in the center, increasing outwards.

Figure 2

Reversing overpropionylation. (A) Overpropionylation in method H 42x can be (partially) reversed by boiling the sample for 1 h or adding HA. Each method is represented by a radar chart showing the average contribution of over‐ (blue), under‐ (yellow) and desired propionylation for seven peptides monitored. Each peptide is located on one angle of the radar chart and each peptide form is represented by another color. The abundance of a specific peptide form is shown on the radius, whereby a conversion rate of 0 is located in the center, increasing outwards. (B) Reaction mechanism of reversing overpropionylation by means of HA. HA is a strong nucleophile which will attack the carbonyl group and thereby induce acyl removal.