2.6. Electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used in two modes: liquid chromatography–mass spectrometry (LC–MS) was used for intact mass analysis while tandem mass spectrometry (MS/MS) was used for peptide sequence analysis. All ESI-MS was undertaken on a Q-ToF Micro mass spectrometer (Waters, Manchester, UK) in positive ion mode. As an additional aid in the interpretation of tandem mass spectra, peptides were isotopically labelled with ¹⁸O by performing proteolytic digestion in a 1 : 1 mixture of light (H₂[¹⁶O]) and heavy (H₂[¹⁸O]) water. Incorporation of a 1 : 1 mixture of [¹⁶O] and [¹⁸O] atoms into the newly formed C-termini of peptides prior to tandem mass spectrometry allowed y-ions to be identified as a sequence of doublets of approximately equal intensity, separated by 2 Da. To confirm and complete the sequence, we repeated the digestions and analysed the samples on a high-resolution instrument with high mass accuracy and resolution for precursor and product ions. For this stage, samples were analysed using a Ultimate 3000 nano system (Dionex/Thermo Fisher Scientific, Hemel Hempstead, UK) coupled with a QExactive mass spectrometer (Thermo Fisher Scientific).