Two-Dimensional Partial Covariance Mass Spectrometry for Macromolecular Sequence Analysis
Prof. Vitali Averbukh, Department of Physics, Imperial College London, UK
We introduce a new type of two-dimensional mass spectrometry (2D-PC-MS) revealing, for the first time, peptide and protein fragment-fragment correlations and leading to the biomolecular sequence reconstruction of unparalleled fidelity.
Two-dimensional covariance mapping spectroscopy was originally developed as a tool for the study of mechanisms of radiation-induced fragmentation of di- and tri-atomic molecules . Until very recently, successful applications of covariance mapping were restricted to atoms and small molecules of up to around 50 Daltons. The main problem arising in applications of covariance mapping to larger molecular species stems from the overwhelming spurious signals of no physical significance induced by fluctuations of experimental parameters. Partial covariance mapping attempts to tackle this problem by taking into account the independently measured parameter fluctuations. However, for macromolecules this becomes unfeasible because of their complex methods of gas-phase delivery and activation that result in a rich manifold of fluctuating experimental parameters, precluding identification of the true physical correlations.
We have recently pioneered the self-correcting partial-covariance spectroscopy which eliminates the need for continuous monitoring of multiple fluctuating experimental parameters enabling covariance mapping of macromolecular decompositions . The self-correcting partial covariance spectroscopy removes the spurious correlations using a single parameter extracted from the spectrum itself – the total ion count (TIC):
𝑝𝐶𝑜𝑣(𝑋,𝑌,𝑇𝐼𝐶) =𝐶𝑜𝑣(𝑋,𝑌)−𝐶𝑜𝑣(𝑋,𝑇𝐼𝐶)𝐶𝑜𝑣(𝑌,𝑇𝐼𝐶) /𝐶𝑜𝑣(𝑇𝐼𝐶,𝑇𝐼𝐶).
The TIC-based self-correcting partial covariance spectroscopy has been shown to be false-positive-free. It leads to successful mapping of the true physical fragment-fragment correlations for decompositions of molecules as large as 103-104 Da. This opens the opportunity for mechanistic studies of macromolecular decompositions using covariance mapping. Moreover, we demonstrate that the two-dimensional partial covariance mass spectrometry (2F-PC-MS) based on the self-correcting partial covariance mapping has an unparalleled analytical capability. 2D-PC-MS enables confident reconstruction of the biomolecular sequences and their covalent modifications in the cases where the standard MS fails as a matter of principle – even at the theoretical infinite mass precision and resolution, as well as full sequence coverage by biomolecular fragmentations. The sequence reconstruction is achieved using the first of its kind proteomic database search engine based entirely on the fragment-fragment correlations , rather than on the individual fragments as in the state-of-the-art software. 2D-PC-MS has been further extended to sequence determination of intact proteins within the “top-down” paradigm .
 L. J. Frasinski, K. Codling and P. A. Hatherly, Science 246, 1029 (1989).  T. Driver, B. Cooper, R. Ayers, R. Pipkorn, S. Patchkovskii, V. Averbukh, D. R. Klug, J. P. Marangos, L. J. Frasinski and M. Edelson-Averbukh, Phys. Rev. X 10, 041004 (2020), see also in Physics Today, DOI:10.1063/PT.6.1.20201023a.  T. Driver, N. Bachhawat, R Pipkorn, L. J. Frasinski, J. P. Marangos, M. Edelson-Averbukh and V. Averbukh, Anal. Chem. 93, 14946 (2021).  T. Driver, V. Averbukh, L. J. Frasinski, J. P. Marangos and M. Edelson-Averbukh, Anal. Chem. 93, 10779 (2021).