Quantitative characterization of histone post-translational modifications using a stable isotope dimethyl-labeling strategy

Abstract

Histone post-translational modifications (PTMs) have been considered to be a major group of important epigenetic marks and to play a critical role in the regulation of chromatintemplated biological processes. To date, novel strategies for the quantification of histone PTMs are still highly desirable. Here, we present an efficient approach to quantitatively characterize histone PTMs using stable isotope dimethyl-labeling coupled with mass spectrometry. At first, all the ε-amino groups of free lysines are derivated by heavy formaldehyde to enable an easy distinction of free lysines from those of naturally occurring lysine-dimethylation upon MS analysis. After tryptic digestion, a second derivatization was applied with heavy and light stable isotope dimethyl-labeling to respectively label the Ntermini of tryptic peptides from different sample sources. The mixture were further identified and quantified by HPLC/MS/MS. This method enables to compare histone PTMs from multiple sample sources and to quantify different PTMs at certain amino acid residues of histone in one single experiment. Thus it is highly attractive for the identification of epigenetic histone marks.

Accelerating the Field of Epigenetic Histone Modification Through Mass Spectrometry–Based Approaches

“…Different derivatization methods have been optimized in several laboratories, from the use of formaldehyde ( 20 ) to maleic anhydride ( 21 ) to propionic anhydride ( 22 ). Sidoli et al .…”

“…The peptides can now be easily resolved by standard reversed-phase chromatography. Stable isotope-labeled anhydrides can be introduced in the second derivatization step after trypsin digestion to label newly generated peptide N termini, allowing accurate quantification and comparison of histone PTMs from two separate conditions in one MS run ( 20 , 22 ).…”

“…Different derivatization methods have been optimized in different laboratories, from the use of formaldehyde (15) to maleic anhydride (16) to propionic anhydride (17). Sidoli et.al tested twelve commercially available anhydrides for histone derivatization and concluded propionic anhydride derivatization as the best to achieve high MS sensitivity and ionization efficiency for histone analysis (18).…”

“…In addition to preventing cleavage at lysine residues, the propionic anhydride derivatization neutralizes the charge, making the resulting peptides less hydrophilic which are easily resolved by standard reversed-phase chromatography. Stable isotope labeled anhydrides can be introduced in the second derivatization step after trypsin digestion to label newly generated peptide N-termini, allowing accurate relative quantification and comparison of histone PTMs from two separate conditions in one MS run (15,17). The protocol for bottom-up analysis has been optimized for histone extraction, MS acquisition method, and data analysis, to provide the most robust and accurate characterization of histone PTMs (14,19,20).…”

Cracking chromatin with proteomics: From chromatome to histone modifications

“…In this condition ArgC is not efficient therefore requiring alternative efficient in-gel digestion methods. Derivatization of lysine residues followed by tryptic digestion represents a very valuable method as it prevents cleavage at lysines, thus promoting an in-gel ArgC-like digestion, peptides with chemical and physical properties optimal for the MS analysis [121][122][123][124][125]. More recently, phenyl isocyanate (PIC) has been also explored for N-terminal derivatization in conjunction with the use of deuterated or propionylated anhydride for the acylation of the lateral ammine group of lysine residues, boosting the identification of very hydrophilic peptides [117,126,127].…”