Charging of Proteins in Native Mass Spectrometry

Abstract

Factors that influence the charging of protein ions formed by electrospray ionization from aqueous solutions in which proteins have native structures and function were investigated. Protein ions ranging in molecular weight from 12.3 to 79.7 kDa and pI values from 5.4 to 9.6 were formed from different solutions and reacted with volatile bases of gas-phase basicities higher than that of ammonia in the cell of a Fourier-transform ion cyclotron resonance mass spectrometer. The charge-state distribution of cytochrome c ions formed from aqueous ammonium or potassium acetate is the same. Moreover, ions formed from these two solutions do not undergo proton transfer to 2-fluoropyridine, which is 8 kcal/mol more basic than ammonia. These results provide compelling evidence that proton transfer between ammonia and protein ions does not limit protein ion charge in native electrospray ionization. Both circular dichroism and ion mobility measurements indicate that there are differences in conformations of proteins in pure water and aqueous ammonium acetate, and these differences can account for the difference in the extent of charging and proton-transfer reactivities of protein ions formed from these solutions. The extent of proton transfer of the protein ions with higher gas-phase basicity bases trends with how closely the protein ions are charged to the value predicted by the Rayleigh limit for spherical water droplets approximately the same size as the proteins. These results indicate that droplet charge limits protein ion charge in native mass spectrometry and are consistent with these ions being formed by the charged residue mechanism. Graphical Abstract ᅟ.

Keywords: Ammonium; Apparent gas-phase basicity; Charged residue mechanism; Charging; Charging mechanism; Circular dichroism; Combined charged residue-field emission model; ESI; Electrospray; Electrospray ionization; Gas-phase basicity; Ion mobility; Mechanism; Native ESI; Native MS; Native electrospray; Native mass spec; Native mass spectrometry; Protein ion charging; Proton transfer; Rayleigh limit; Salts.

Figure 1

(a) CD spectra of 10 μM carbonic anhydrase and (b) 10 μM cytochrome c in pure water (solid blue line), 10 mM ammonium acetate (green dotted line), and 10 mM potassium acetate (purple dashed line). (c) Collision cross sections of 10 μM cytochrome c ions (pink) and carbonic anhydrase ions (blue) and formed from 10 mM ammonium acetate (square, diamond, respectively) or pure water (triangle, circle, respectively) as a function of charge state. (d) Arrival time distributions of cytochrome c 8+ ions and carbonic anhydrase 12+ ions formed from water (blue) or 10 mM ammonium acetate (green)

Figure 2

Mass spectra of concanavalin A dimer ions formed from 10 mM ammonium acetate after reaction with either 2-fluoropyridine or pyridine for (a) 0 and (b) 120 s

Figure 3

Mass spectra of 10 μM cytochrome c ions formed from 10 mM ammonium acetate and 10 mM potassium acetate reacted with 2-fluoropyrine for 0 and 120 s

Figure 4

Z_max (purple square), Z_av (pink triangles), Z_R(CCS) (blue circles) for protein ions formed from aqueous ammonium acetate (filled) or pure water (open) relative to Z_R (green line) as a function of molecular weight. Z_R represents the maximum number of charges on a protein predicted using the Rayleigh limit for a droplet the same size as a spherical protein with a density of 1.0 g/cm³. Z_R(CCS) represents the maximum number of charges predicted using the Rayleigh limit for a spherical droplet with the same radius as the CCS_av for the protein ions.

Figure 5

(a) The percent Z_max (circle) and Z_av (square) of Z_R, for protein ions formed from aqueous ammonium acetate (filled markers) or pure water (open markers) prior to reaction with a base v. the decrease in Z_max (circle) and Z_av (square) after 120 s reaction with DPA. (b) fraction of basic residues, (c) molecular weight (d) number of basic residues (e) isoelectric point (pI) for each protein as a function of the decrease in Z_max (circle) and Z_av (square) after 120 s reaction with DPA. (i) corresponds to cytochrome c, (ii) myoglobin, (iii) carbonic anhydrase, (iv) concanavalin A dimer and (v) holo-transferrin ions, respectively.