Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

Abstract

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

Figure 1

Charging of EXG variants in ESI-MS. (A) Wt EXG_WT and the chargeless variant EXG_QQQW have highly similar three-dimensional structures. Protein surfaces are rendered according to Coulombic potential at pH 7.0, showing the effect of four replacements (K28Q, D36Q, R68Q, and H90W) on the surface charge of EXG_QQQW. (B) ESI-Mass spectra of a 1:3 mixture of EXG_WT and EXG_QQQW show identical charge state envelopes for both variants in positive (top) and negative (bottom) ionization modes.

Figure 2

Folded states of EXG_WT and EXG_QQQW in solution and in the gas phase. (A) CD spectroscopy in the far-UV region shows near-identical secondary structures in 10 mM sodium phosphate buffer pH 7. In 50% acetonitrile, EXG_QQQW, but not EXG_WT, converts to random coil. Greyed-out parts of the curves exhibited HT voltages above 700 V, indicating increased spectral noise. (B) ESI-MS shows a slight increase of the average charge to 5.7 for both proteins sprayed from 50% acetonitrile. (C) In 100 mM AmAc, the 6+ charge states of EXG_WT and EXG_QQQW have similar CCS distributions. (D) In 50% acetonitrile, the CCS distribution for EXG_QQQW shows an extra peak around 1550 Ų, indicating a second, more unfolded population.

Figure 3

Charging and gas-phase stabilities of WT and supercharged GFP variants. (A) Coulombic surface potentials predicted for the three GFP variants. Structural models were generated using Rosetta; the His₆ tag is omitted for clarity. (B) Mass spectra of GFP_Ac, GFP_WT, and GFP_Bas show near-identical CSDs dominated by the 9+ and 10+ charge states. (C) CIU profiles of the 9+ charge state of each variant indicate that GFP_Bas has a higher resistance to unfolding than GFP_WT and GFP_Ac.

Figure 4

Changing the location of individual lysine residues affects the gas-phase conformation of TTHA1718 variants. (A) ESI mass spectra of TTHA_K5E in ammonium acetate show a narrow CSD of 4+ and 5+ ions and in dH₂O show a broad CSD ranging from 4+ to 9+. (B) The locations of the 9 lysine residues are indicated on the Coulombic surface representation of WT TTHA1718 (PDB ID 2ROE). (C) The CCS distributions of the 5+ charge states of TTHA_K5E, TTHA_K20E, TTHA_K30E, and TTHA_K61E sprayed from AmAc at a trap voltage of 5 V show a small native-like population for TTHA_K20E and TTHA_K30E, whereas TTHA_K5E and TTHA_K61E are mostly unfolded. The 8+ charge states from dH₂O, which can be considered mostly unfolded, reveal variant-specific differences in their CCS distributions. The dashed line indicates the theoretical CCS of TTHA1718.