Ion Coalescence in Fourier Transform Mass Spectrometry: Should We Worry about This in Shotgun Proteomics?

Coupling of motion of the ion clouds with close m/z values is a well-established phenomenon for ion-trapping mass analyzers. In Fourier transform ion cyclotron resonance mass spectrometry it is known as ion coalescence. Recently, ion coalescence was demonstrated and semiquantitatively characterized for the Orbitrap mass analyzer as well. When it occurs, the coalescence negatively affects the basic characteristics of a mass analyzer. Specifically, the dynamic range available for the high resolving power mass measurements reduces. In shotgun proteomics, another potentially adverse effect of ion coalescence is interference of the isotopic envelopes for the coeluting precursor ions of close m/z values, subjected to isolation before fragmentation. In this work we characterize coalescence events for synthetic peptide mixtures with fully and partially overlapping ¹³C-isotope envelopes, including pairs of peptides with glutamine deamidation. Furthermore, we demonstrate that fragmentation of the otherwise coalesced peptide ion clouds may remove the locking between them owing to the total charge redistribution between more ion species in the mass spectrum. Finally, we estimated the possible scale of the coalescence phenomenon for shotgun proteomics by considering the fraction of coeluted peptide pairs with the close masses using literature data for the yeast proteome. It was found that up to one-tenth of all peptide identifications with the relative mass differences of 20 ppm or less in the corresponding pairs may potentially experience the coalescence of the ¹³C-isotopic envelopes. However, sample complexity in a real proteomics experiment and precursor ion-signal splitting between many channels in tandem mass spectrometry drastically increase the threshold for coalescence, thus leading to practically coalescence-free proteomics based on Fourier transform mass spectrometry.