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

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While mass spectrometry (MS) is the favored analytical technique for the primary structures of proteins and their post-translational modifications (PTMs), the combinatorial isomers that commonly co-occur necessitate a more advanced approach.

A study led by researchers at Imperial College London in the UK devised a two-dimensional partial covariance mass spectrometry (2D-PC-MS) platform for the evaluation of cofragmented combinatorial isomers that improves upon standard MS by not requiring a priori knowledge of fragment mass spectra of any individual isomer (1). The added method of marker ion correlations within this approach, the research team said, showed the potential to be expanded to analysis of other biopolymers.

Cofragmented combinatorial isomers refer to a specific phenomenon observed in mass spectrometry-based analysis of complex mixtures. In this context, combinatorial isomers are molecules with identical molecular formulae but different structural arrangements. During fragmentation in the mass spectrometer, these isomers produce identical fragment ions, leading to overlapping mass spectra. Distinguishing and resolving cofragmented combinatorial isomers can be challenging but is essential for accurate identification and quantification in metabolomics and other analytical applications.

Recently published in the Journal of the American Society for Mass Spectrometry, this report builds on previous studies focusing on histone proteins, the best-known system of combinatorial isomers, which are biomolecules that have the same chemical sequence and same number of identical covalent modifications, though distributed differently across modification sites (1). Post-translational modifications (PTMs) to histone tails are thought to act sequentially or in concert to signal various biological functions. This study refers to that phenomenon as the “histone code,” which can perpetuate cell differentiation and reprogramming as well as embryonic development, resulting in human health effects on aging, certain cancers, and neurodegenerative disorders.

Numerous shortcomings in MS-based histone code analysis are described by the researchers, including an attempt to exploit oft-subtle differences in the signal intensities of fragments, and an alternative use of spectral libraries that the authors described as inherently ambiguous because experimental conditions may fluctuate (1). Even tandem mass spectrometry (MS/MS) cannot always overcome coelution and, subsequently, coisolation caused by small differences in the physical properties of positional isomers. Certain modern software programs listed by the researchers are also based on individual fragment ions, and are of no assistance.


A marker ion correlation method—which the researchers distinguished from marker ions themselves—does not require preliminary information about individual isomer behavior with respect to MS/MS (1). Full sets of marker ions are not accessible even with MS/MS, according to this study, because of m/z degeneracy of the fragment ions produced by cofragmented combinatorial isomers; this precludes a thorough identification of the isomers under analysis. On the other hand, a dedicated database search engine and outline for the 2D-PC-MS approach, namely physical principle and measurement procedures and data processing, have been demonstrated in previous reports.

While particular attention was paid to mixtures of the synthetically diacetylated positional isomers of histone H4 fragment 4–17, the in silico study in general resulted in five times more cofragmented combinatorially acetylated tryptic peptides, and three times more combinatorially modified Glu-C peptides, being unambiguously identified in human histones using marker ion correlations compared to standard MS (1). As the researchers explained, diacetylated histone H4 peptides had always presented a unique challenge to state-of-the-art MS. But with the 2D-PC-MS method used in this instance, there lies newfound potential to link pairs of individual fragments that not otherwise isomer-specific to a single isomeric sequence. The researchers expressed hope that their work might lead to better analysis of methylated oligonucleotides, just to use one example, as the method gains in reputation and effectiveness.


(1) Driver, T.; Pipkorn, R.; Averbukh, V.; Frasinski, L.J.; Marangos, J.P.; Edelson-Averbukh, M. Identification of Cofragmented Combinatorial Peptide Isomers by Two-Dimensional Partial Covariance Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2023, 34 (7), 1230–1234. DOI: 10.1021/jasms.3c00111