Identification of Cofragmented Combinatorial Peptide Isomers by Two-Dimensional Partial Covariance Mass Spectrometry

Combinatorial post-translational modifications (PTMs),such asthose forming the so-called "histone code", have beenlinked to cell differentiation, embryonic development, cellular reprogramming,aging, cancers, neurodegenerative disorders, etc.Nevertheless, a reliable mass spectral analysis of the combinatorialisomers represents a considerable challenge. The difficulty stemsfrom the incompleteness of information that could be generated bythe standard MS to differentiate cofragmented isomeric sequences intheir naturally occurring mixtures based on the fragment mass-to-chargeratio and relative abundance information only. Here we show that fragment-fragmentcorrelations revealed by two-dimensional partial covariance mass spectrometry(2D-PC-MS) allow one to solve the combinatorial PTM puzzles that cannotbe tackled by the standard MS as a matter of principle. We introduce2D-PC-MS marker ion correlation approach and demonstrate experimentallythat it can provide the missing information enabling identificationof cofragmentated combinatorially modified isomers. Our insilico study shows that the marker ion correlations can beused to unambiguously identify 5 times more cofragmented combinatoriallyacetylated tryptic peptides and 3 times more combinatorially modifiedGlu-C peptides of human histones than is possible using standard MSmethods.