Enhancing signal and mitigating up-front peptide fragmentation using controlled clustering by gas-phase modifiers.

Up-front CID fragmentation is a phenomenon where molecular ions are activated and fragment as they enter the atmosphere-to-vacuum region of the mass spectrometer, and consequently can complicate the mass spectra and their analysis. This phenomenon can be minimized by controlling the voltages on lens/optic elements where ions are sampled from the atmospheric region, but this approach can also have a negative effect on overall ion sensitivity. In this study, we introduce gas-phase modifiers (acetonitrile, acetone, cyclohexane, water, and methanol) to the curtain gas to mitigate up-front CID fragmentation. These modifiers cluster with incoming ions, increasing the energy barrier to fragmentation and consequently reducing the complexity of mass spectra. The clustering is monitored by differential mobility spectrometry-mass spectrometry (DMS-MS) and precursor mass spectrum-scanning. Unlike typical singly charged species, peptide ion-modifier clusters were found to survive through the atmosphere-to-vacuum interface of the mass spectrometer, showing that highly charged peptides cluster most strongly with acetonitrile and acetone. In addition, when peptides cluster with acetonitrile, they produce a large increase in signal intensity for the most highly charged and fragile ions. This results in a significant reduction, up to 90% with some modifiers, in up-front CID fragmentation for these fragile highly charged peptides, increasing the overall analytical sensitivity and decreasing the limits of detection by up to 82% depending on the analyte. The proposed technique has no significant detrimental effect on the peptide mass fingerprinting of a BSA or mAb protein digest, but it does reduce the amount of redundant and data-deficient spectra needed to produce adequate sequence coverage using information-dependent acquisition methods by ~ 40%. We propose that this technique could have a benefit in the fields of proteomics and peptidomics where up-front CID fragmentation and chemical noise routinely mask targets of biological importance. Graphical abstract.