To characterise sequence and post-translational modifications of proteins, the molecular ion and fragment ion data that is obtained by activating an intact protein ion inside a mass spectrometer (MS) can be significantly more discerning than that obtained for peptides formed by protein digestion. The primary advantage of intact protein MS is that degradation products, sequence variants, and different combinations of post-translational modifications can be distinguished. However, complete characterisation of intact protein ions that are larger than ca. 10 kDa is challenging primarily because ions in relatively low charge states do not readily fragment unlike those in higher charge states. Here, we report our discoveries that (i) proteins can be supercharged beyond the theoretical limit to protein charging in electrospray ionisation (ESI), which is based on gas-phase proton-transfer reactivity, and (ii) these ions that are formed by “extreme” supercharging fragment significantly more readily than lower charge states in ion-electron recombination experiments (electron capture dissociation, ECD; e.g., greater than 85% of 259 inter-residue sites can be identified in a single MS experiment for bovine serum albumin). The first results from ultraviolet laser induced photodissociation of extremely supercharged protein ions will also be reported.
“Extreme” protein supercharging was discovered by adding butylene carbonate (and structurally related analogue molecules) to ESI solutions, which resulted in the formation of protein ions in significantly higher charge states than have been reported by use of other methods/additives. ECD data was obtained using a Thermo 7 T LTQ-FT/ICR MS (UNSW). Ultraviolet laser induced photodissociation tandem mass spectrometry measurements of intact protein ions were performed using a Orbitrap Q Exactive Plus (U Melbourne), which has been equipped to allow m/z-selected ions to be irradiated with a single pulse of 193 nm photons (6.4 eV).
“Extreme” supercharging of protein ions is useful for significantly improving the sequence characterisation of intact proteins by top-down MS.