Analysis of protein mixtures by electrospray mass spectrometry: Effects of conformation and desolvation behavior on the signal intensities of hemoglobin subunits

Hemoglobin (Hb) is a tetrameric noncovalent complex consisting of two ␣-and two ␣ ␤-globin ␤ chains each associated with a heme group. Its exact assembly pathway is a matter of debate. Disorders of hemoglobin are the most common inherited disorders and subsequently the molecule has been extensively studied. This work attempts to further elucidate the structural properties of the hemoglobin tetramer and its components. Gas-phase conformations of hemoglobin tetramers and their constituents were investigated by means of traveling-wave ion mobility mass spectrometry. Sickle (HbS) and normal (HbA) hemoglobin molecules were analyzed to determine whether conformational differences in their quaternary structure could be observed. Rotationally averaged collision cross sections were estimated for tetramer, dimer, apo-, and holo-monomers with reference to a protein standard with known cross sections. Estimates of cross section obtained for the tetramers were compared to values calculated from X-ray crystallographic structures. HbS was consistently estimated to have a larger cross section than that of HbA, comparable with values obtained from X-ray crystallographic structures. Nontetrameric species observed included apo-and holo-forms of ␣-and ␣ ␤-monomers and ␤ heterodimers; ␣-and ␣ ␤-monomers in both apo-and holo-forms were found to have similar ␤ cross sections, suggesting they maintain a similar fold in the gas phase in both the presence and the absence of heme. Heme-deficient dimer, observed in the spectrum when analyzing commercially prepared Hb, was not observed when analyzing fresh blood. This implies that holo-␣-apo-␣ ␤ is not an essential intermediate within the The typical electrospray ionization (ESI) mass spectrum of a protein consists of an envelope of peaks attributed to a series of multiply charged gas-phase ions that can indicate the stability and compactness of its structure in the gas phase [4,5]. Multiply charged ions are produced by proton attachment, predominantly to exposed basic sites on the protein [6], and those of lowest charge are thought to be most representative of native structure [7]. A highly compact protein would have fewer exposed basic sites than those of an unfolded conformation of the same protein and thus would accept fewer charges [4,8].Ion mobility spectrometry is a shape-selective technique, based on the time taken for an ion to traverse a mobility cell containing an inert gas under the influence of a weak electric field [9]. This time is related to the rotationally averaged collision cross section, mass, and charge of the ion. The coupling of ion mobility separation with mass spectrometry has provided a powerful method for the analysis of complex mixtures and for the determination of molecular structure [10].Ion mobility mass spectrometry has emerged as a complementary technique to the well-established methods of X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy for three-dimensional protein structure analysis [11]. There is now substantial evidence to support...

Section: Hemoglobin Tetramer Analysissupporting

confidence: 90%

Section: Hemoglobin Tetramer Analysismentioning

confidence: 99%

Probing hemoglobin structure by means of traveling-wave ion mobility mass spectrometry

Scarff

Patel

Thalassinos

et al. 2008

“…Because the relative abundances of these species depend strongly on the pressure in the ion guide, it is difficult to draw conclusions about the relative abundances of these species in solution from the mass spectra, but it seems the levels of dimers and monomers are greater than expected from the equilibrium solution dissociation of hemoglobin. Recent studies using freshly prepared hemoglobin have shown that the presence of ␤-monomers in the ESI ␤ spectra (Figure 1a-c) are likely artifacts caused by the lyophilization process used to produce commercial hemoglobin [35,51]. Possibly in these experiments we see higher levels of dimers and monomers than expected because the hemoglobin is oxidized [35], the solutions contain 10% methanol, and multimers can dissociate in the electrospray and ion sampling processes.…”

Section: Mass Spectra Of Hemoglobinmentioning

confidence: 87%

“…At ⌬V OS ϭ 200 V, the tetramer ions are the most abundant, while the dimers dissociate by losing heme to form [31,33,35,42,51]. When ⌬V OS is increased to 200 V, the widths of the multimeric peaks decrease significantly.…”

Section: Cid Of Gas-phase Hemoglobinmentioning

confidence: 99%

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

Wright

Douglas

2009

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...

“…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].…”

Section: Mass Spectra Of Hemoglobinsmentioning

confidence: 79%

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

Kang

Douglas

2011

Hemoglobin (Hb) (α 2 β 2 ) is a tetrameric protein-protein complex. Collision cross sections, hydrogen exchange levels, and tandem mass spectrometry have been used to investigate the properties of gas-phase monomer, dimer, and tetramer ions of adult human hemoglobin (Hb A, α 2 β 2 ), and two variant hemoglobins: fetal hemoglobin (Hb F, α 2 γ 2 ) and sickle hemoglobin (Hb S, α 2 β 2 , E6V[β]). All three proteins give similar mass spectra. Monomers of Hb S and Hb F have similar cross sections, ca. 10% greater than those of Hb A. Cross sections of dimer ions of Hb S are 11% greater than those of Hb A and 6% greater than those of Hb F. Tetramers of Hb S are 13% larger than tetramers of Hb A or Hb F. Monomers and dimers of all three Hb have similar hydrogen-deuterium exchange (HDX) levels. Tetramers of Hb S exchange 16% more hydrogens than Hb A and Hb F. In tandem mass spectrometry, monomers of Hb S and Hb F require ca. 10% greater internal energy for heme loss than Hb A. Dimers (+11) of Hb A and Hb S dissociate to monomers with asymmetrical charge division; dimers of Hb F (+11) dissociate with nearly equal charge division. Tetramer ions dissociate to monomers and trimers, unlike solution Hb, which dissociates to dimers. The most stable dimers are from Hb S; the most stable tetramers from Hb F. The results with Hb S show that a single mutation in the β chain can change the physical properties of this gas-phase protein-protein complex.