All-ions laser induced dissociation (AI-LID) in the C-trap of a Q Exactive: Data independent identification and quantification directly from ultra-high resolution differential mass spectra

Abstract

An analytical method is demonstrated which allows to follow a data independent, all ion fragmentation (AIF) acquisition procedure, but to fall back on a standard and generic data-dependent acquisition (DDA) analysis procedure for the data treatment: searching databases with individual precursor ions and fragments lists. This method is implemented with photo-activation (LID) in the visible range which allows extreme specificity and sensitivity, and is demonstrated on Cysteine-containing tryptic peptides grafted with a chromophore. This proof of concept relies on the efficient implementation of LID directly in the C-trap of a ThermoScientific QExactive mass spectrometer during ion accumulation, before full MS detection. Laser irradiation turned alternatively ON and OFF after each full MS allows to build differential mass spectra in which non-dissociating ions are cancelled out while fragmenting precursors and fragments show up with opposite signs, which enables to build precursor/fragments lists for database search. Protein identification was demonstrated with remarkably low false discovery rates. Relative quantification was also demonstrated by integration of precursor and fragment ion elution peaks after differential analysis and limits of quantifications were demonstrated down to 11 ng of proteins injected. This mixed approach takes the best of both worlds – the deep proteome coverage of DIA, with the exhaustive fragmentation of all precursor ions, and the reliability and ease of use of classical DDA database search, relieving the need for the initial constitution of a spectral library.