Abstract
Histone post-translational modifications (hPTMs) are epigenetic marks that strongly affect numerous processes, including cell cycling and protein interactions. They have been studied by both antibody- and MS-based methods for years, but the analyses are still challenging, mainly because of the diversity of histones and their modifications arising from high contents of reactive amine groups in their amino acid sequences. Here, we introduce use of trimethylacetic anhydride (TMA) as a new reagent for efficient histone derivatization, which is a requirement for bottom–up proteomic hPTM analysis. TMA can derivatize unmodified amine groups of lysine residues and amine groups generated at peptide N-termini by trypsin digestion. The derivatization is facilitated by microwave irradiation, which also reduces incubation times to minutes. We demonstrate that histone derivatization with TMA reliably provides high yields of fully derivatized peptides and thus is an effective alternative to conventional methods. TMA afforded more than 98% and 99% labeling efficiencies for histones H4 and H3, respectively, thereby enabling accurate quantification of peptide forms. Trimethylacetylation substantially improves chromatographic separation of peptide forms, which is essential for direct quantification based on signals extracted from MS1 data. For this purpose, software widely applied by the proteomics community can be used without additional computational development. Thorough comparison with widely applied propionylation highlights the advantages of TMA-based histone derivatization for monitoring hPTMs in biological samples.
Highlights
Microwave-assisted labeling of histones using trimethylacetic anhydride (TMA) for bottom–up proteomics. TMA enables discrimination of isobaric peptides by improved chromatographic behavior. TMA facilitates histone quantification from MS1-level spectral information. TMA provides excellent performance for monitoring histone post-translational modification (hPTM) pattern in the tissues
We introduce use of trimethylacetic anhydride (TMA) as a new reagent for efficient histone derivatization, which is a requirement for bottom–up proteomic hPTM analysis
The overall acetylation trend was similar to that observed in TMA-treated samples, but detailed quantification of propionylated peptide forms was hindered by frequent presence of coeluting peaks of isobaric peptides in the MS1-level spectra and missing identification of certain modified sites (e.g., H3K56ac, H3K79ac, H3K122ac, H4K31ac, H4K59ac, H4K77ac, H4K79ac, and H4K91ac)
Summary
In Brief The discrimination of isobaric peptides represents a common challenge in histone characterization. Growing evidence that histone epigenetic marks participate in various physiological and pathological processes has prompted further development of MS-based strategies for hPTM characterization [13,14,15] These include chemical derivatization of amine groups in histone sequences as a useful strategy for preparing samples for bottom–up LC–MS/MS analysis [16]. In the study presented here, we established a procedure for microwave-assisted labeling of histones prior to bottom–up LC–MS/MS analysis involving use of trimethylacetic anhydride (TMA; known as pivalic anhydride) for lysine derivatization This provided substantial advantages for histone quantification using MS1-level spectral information. For an impartial comparison of the TMA-based procedure, we assessed the performance (using the same parameters) of the widely applied propionylation for labeling the same samples
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