Improved Protein Identification in Native Ambient Mass Spectrometry by the Integration of Proton Transfer Charge...

Here, we demonstrate PTCR-HCD MS3 as a tool for improving protein identification in native ambient mass spectrometry (NAMS). NAMS involves the direct analysis of tissue sections with no or little sample preparation and no (solution-phase) separation postsampling. NAMS samples are therefore highly complex, comprising proteins, salts, and other biomolecules, and NAMS spectra are highly congested. PTCR MS has emerged as a useful strategy in top-down proteomics. Most commonly, it is applied as an adjunct MS2 step, providing molecular weight information to support sequence information provided by collision-based MS2 or it is applied as an MS3 step to simplify collision, photon, or electron-based MS2 by reducing m/z overlap of product ions. In this work, we show that the selection of charge-reduced PTCR product ions for subsequent HCD reduces the incidence of chimeric fragmentation spectra and improves confidence in protein identification in NAMS samples from mouse spinal cord, rat kidney, and cell-line-derived xenograft tissue. As well as removing interference from overlapping protein species, we show that PTCR-HCD MS3 also removes interference from overlapping lipid species which can similarly affect confident protein identification. The approach also allows identification of proteins that are masked at the MS1 level but are revealed following PTCR.