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

Abstract

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...

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

“…These results are based on the BharshÛ (Figure 2a). The measured Ω values agree with experimental literature data to within 2% or better [3,18,65,70]. Most importantly, Figure 4 demonstrates that virtually identical data are obtained when employing regular ESI or nanoESI.…”

“…In the case of Hb the calculated Ω values are 5% (EHSS) and 9% (PA_CORR) smaller than the experimental values. It is unlikely that this mismatch reflects a calibration artifact because (1) the t d of Hb is well within the range covered by calibrant ions, and (2) the measured Ω agrees closely with the literature [65]. In contrast to the monomeric proteins discussed above, Hb therefore appears to be slightly more expanded in the gas phase than in the crystal.…”

“…Mass spectra obtained under these conditions are dominated by low charge states (Figure 3) [68]. A lack of heme loss from hMb [69], as well as the dominance of intact Hb tetramers [48,65] confirm the gentle nature of the experiments. Consistent with recent observations [48], very similar charge state distributions were obtained for nanoESI and regular ESI.…”

“…The exact hard sphere scattering (EHSS) algorithm produces values that are more reliable (referred to as Ω EHSS ) [18]. The low computational cost of the EHSS algorithm has made it the method of choice for many practitioners [3,18,23,65]. Another common approach is the use of rescaled PA data (Ω PA_CORR ), which have been shown to agree well with experimental values [29,56,66].…”

Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: An investigation into eukaryotic initiation factors

“…Whether all proteins remain properly folded or misfolded continues to be a debatable point, and is quite likely to depend on the tertiary structure of each individual protein studied. It has been shown, however, that under carefully controlled acquisi- tion conditions the CCS of the lowest detected charge state will fall within the boundaries of the theoretically calculated values using the PA and EHSS methods of theory [21,34]. Thus the current method of analyzing the k subunit holds true to previous experiments.…”