Gas-phase H/D exchange and collision cross sections of hemoglobin monomers, dimers, and tetramers

Abstract

The conformations of gas-phase ions of hemoglobin, and its dimer and monomer subunits have been studied with H/D exchange and cross section measurements. During the H/D exchange measurements, tetramers undergo slow dissociation to dimers, and dimers to monomers, but this did not prevent drawing conclusions about the relative exchange levels of monomers, dimers, and tetramers. Assembly of the monomers into tetramers, hexamers, and octamers causes the monomers to exchange a greater fraction of their hydrogens. Dimer ions, however, exchange a lower fraction of their hydrogens than monomers or tetramers. Solvation of tetramers affects the exchange kinetics. Solvation molecules do not appear to exchange, and solvation lowers the overall exchange level of the tetramers. Cross section measurements show that monomer ions in low charge states, and tetramer ions have compact structures, comparable in size to the native conformations in solution. Dimers have remarkably compact structures, considerably smaller than the native conformation in solution and smaller than might be expected from the monomer or tetramer cross sections. This is consistent with the relatively low level of exchange of the dimers. . Electrospray ionization has allowed the study of protein ions in the gas phase, free of water. In the absence of water, monomeric proteins can adopt compact conformations similar to the solution conformations, but also extended conformations far removed from the native state [2,3]. Much of the current understanding of the "size" of protein ions in the gas phase comes from physical methods of measuring collision cross sections using ion mobility spectrometry at pressures of a few Torr [4 -7], at atmospheric pressure [8], or at pressures of ca. 1 ϫ 10 Ϫ3 Torr [7,9,10]. Measurements of ion kinetic energy loss in triple quadrupole systems have also been used to determine collision cross sections of proteins [11][12][13] and protein-ligand complexes [11,14,15]. Chemical probes of protein conformation have been used as well, with much of the work using gas-phase hydrogen deuterium exchange (H/Dx) of trapped ions, either in linear [16 -19] or 3D [20] quadrupole ion traps, or ion cyclotron resonance (ICR) mass spectrometers [21][22][23][24][25].The study of the structure of monomeric proteins in the gas phase has been extended to assemblies containing more than one protein. Ion mobility measurements of complex macromolecular protein complexes at atmospheric pressure [8] or at a pressures of ca. 1 ϫ 10 Ϫ3 Torr have shown some success [9,10]. The resolution of these experiments, ca. 5-20, is lower than more conventional mobility measurements at pressures of a few Torr.Protein-protein and protein-ligand complexes can be studied by gas-phase H/Dx of trapped ions. The pressures of deuterating reagent in linear trap experiments can be ϳ5 ϫ 10 Ϫ3 Torr, ca. 10 3 times greater than used in 3D traps [20], 10 1 to 10 2 times greater than used in mobility experiments [26,27], and 10 3 to 10 4 times greater than in ICR experiments [21][22][2...

Gas-Phase Ions of Human Hemoglobin A, F, and S

“…Lowering the Hb concentration to 10 μM and changing the solvent to 10% ACN, which slightly destabilizes the protein, increases the levels of monomer ions (apo-and holo-) and dimer ions (Figure 1c and d), as in our previous study [17], making measurement of the properties of monomer and dimer ions possible. In this case, the variant β chains of Hb S and the γ chains of Hb F can be seen but with much lower intensity than the α chains, similar to Hb A [18,40,45].…”

“…LIT-TOF System and Gas-Phase HDX HDX experiments were performed with a home-made linear quadrupole ion trap reflectron time-of-flight mass spectrometer system (LIT-TOF), as described previously [18,[29][30][31][32]. Protonated ions generated by pneumatically assisted ESI (5 kV), pass through an aperture (5 mm diameter) in a curtain plate (1000 V), a dry nitrogen curtain gas (~2 L/min), an orifice (0.25 mm diameter, 220 V), a skimmer (0.75 mm diameter, 20 V), and enter a chamber with two consecutive radio frequency only quadrupoles, Q0 (DC offset=15 V) and Q1 (DC offset=10 V).…”

“…Typical trapping conditions: 50 ms injection (Q0/Q1=−5 V, L1=60 V), 0-10,000 ms trap (Q0/Q1=40 V, L1=60 V), 20 ms detection (Q0/Q1=40 V, L1=−15 V), and 50 ms drain (Q0/Q1=−5 V, L1=−15 V). After trapping, ions pass through a stack of four focusing lenses (L1-L4), then are mass analyzed with a reflectron-TOF and detected by a dual microchannel plate [18]. A solution of CsI was used for mass calibration.…”

“…1.02 for +15 ions to 1.07 for +18 ions [16]. An alternative approach to determining cross sections is to measure ion axial kinetic energy losses with a triple quadrupole mass spectrometer [17,18]. In a recent study of human Hb A, we showed that oxidation of methionine and cysteine residues on the β-chain, and lyophilization of Hb, change the appearance of Hb mass spectra but do not influence the cross sections of gas-phase Hb ions [17].…”

“…With Hb, HDX can be done in solution prior to injection of Hb into an ESI source [19] or directly in the gas phase with trapped ions exposed to D 2 O vapor [18]. The mechanism of solutionphase HDX is well established [20].…”