Breaking the Histone Code with Two-Dimensional Partial Covariance Mass Spectrometry

Combinatorial post-translational modifications (PTMs) of proteins, such as histones, govern cell differentiation and organismal development, and are widely thought to play a key role in aging, development of cancers, neurodevelopmental disorders, neurodegenerative and other diseases. Nonetheless, current understanding of the precise biological function of different modification patterns is limited by the difficulty of mass spectrometry to identify and quantify different combinatorial isomers in their mixtures as they naturally occur. This profound difficulty is a result of the fundamental incompleteness of the information contained in a one-dimensional mass spectrum featuring the mass-to-charge ratios and relative abundances of the individual peptide fragments. Here we demonstrate that the fragment-fragment correlations revealed by the recently developed two-dimensional partial covariance mass spectrometry (2D-PC-MS) allow one to solve the combinatorial PTM puzzles that cannot be tackled by the standard mass spectrometry as a matter of principle. We introduce the concept of 2D-PC-MS marker ion correlations and show that they can provide the missing PTM location information that enables identification of co-fragmentated combinatorially modified isomers in their mixtures. We demonstrate experimentally the use of the marker ion correlations to fully analyze and resolve mixtures of doubly acetylated histone H4 peptides, a problem that was previously branded “mathematically impossible”. Our accompanying comprehensive in silico study reveals that the marker ion correlations can be used to unambiguously identify five times more combinatorially modified tryptic peptides and three times more combinatorially modified Glu-C peptides of human histones than is possible using the standard MS/MS.