Polydispersity of a mammalian chaperone: mass spectrometry reveals the population of oligomers in alphaB-crystallin

Abstract

The quaternary structure of the polydisperse mammalian chaperone alphaB-crystallin, a member of the small heat-shock protein family, has been investigated by using electrospray mass spectrometry. The intact assemblies give rise to mass spectra that are complicated by the overlapping of charge states from the different constituent oligomers. Therefore, to determine which oligomers are formed by this protein, tandem mass spectrometry experiments were performed. The spectra reveal a distribution, primarily of oligomers containing 24-33 subunits, the relative populations of which were quantified, to reveal a dominant species being composed of 28 subunits. Additionally, low levels of oligomers as small as 10-mers and as large as 40-mers were observed. Interpretation of the tandem mass spectral data was confirmed by simulating and summing spectra arising from the major individual oligomers. The ability of mass spectrometry to quantify the relative populations of particular oligomeric states also revealed that, contrary to the dimeric associations observed in other small heat-shock proteins, there is no evidence for any stable substructures of bovine alphaB-crystallin isolated from the lens.

Fig. 1.

Survey of the changes in mass spectra of consecutive fractions of αB-crystallin separated by size-exclusion chromatography. (A) Elution profile of bovine αB-crystallin refolded on column from 6 M urea into 200 mM ammonium acetate. The fractions indicated were collected and kept on ice before MS analysis. mAU, milliabsorbance units. (B) Electrospray mass spectra of fractions 23–28 from A. A shift in the distribution of ions on the m/z scale, from high to low values, was observed across the elution profile, indicating a significant decrease in the oligomeric size of αB-crystallin between fractions at the onset and tail of the peak.

Fig. 2.

(A) Electrospray mass spectrum of the combined fractions of bovine αB-crystallin from size-exclusion chromatography. Signal is observed between 7,000 and 15,000 m/z, with the most intense peaks at ≈10,000 m/z. These peaks are not due to a charge-state series of a single oligomer but rather to the overlap of several such series from different-sized oligomers. The spectrum was acquired at a voltage applied to the collision cell of 4 V. (B) Mass spectra obtained for a range of collision energies demonstrate that αB-crystallin oligomers undergo multiple loss of monomers, from one at a voltage applied to the collision cell of 120 V up to three at 200 V. (C) Expansion of the monomer region at voltages applied to the collision cell of 120, 160, and 200 V. The average charge state of the dissociated monomers was observed to decrease from 18+ at 120 V to 16+ at 160 V and 15+ at 200V.

Fig. 3.

MS/MS of the overlapping components in the isolated peak at 10,040 m/z (Fig. 2 A) resulted in dissociation of the oligomers into highly charged monomers at low m/z and various stripped oligomers at high m/z values. Spectrum showing a summation of the acquisitions for a series of voltages applied to the collision cell of 80–200 V. Overlaid is a schematic showing a possible pathway for the multiple loss of monomers from a representative oligomer and the resultant mass and charge asymmetry during the gas-phase CID. The schematic shows a sequential pathway; however, we cannot rule out the simultaneous dissociation of two and three monomers from a single oligomer.

Fig. 4.

(A) Expansion of the region of the spectrum corresponding to oligomers with n – 2 subunits obtained by MS/MS analysis at a voltage applied to the collision cell of 130 V. Peaks corresponding to the charge states of doubly stripped oligomers with different numbers of subunits (n – 2) are observed. The overlapping peak at 20,080 m/z results from oligomers carrying the same number of charges as subunits. The groups of peaks at lower m/z than this central peak result from oligomer populations carrying more than one charge per subunit (n – 1, n, and n + 1), whereas those at higher m/z arise from oligomers carrying less than one charge per subunit (n – 3, n – 4, and n – 5). The gray spectrum represents the sum of the simulated spectra presented in B. (B) A theoretical deconvolution of the spectrum demonstrates how several different-sized stripped oligomers combine to give the observed spectrum (A). Spectra for different-sized doubly stripped oligomers [23 ≤ (n – 2) ≤ 30] were simulated from the theoretical m/z values for each oligomer ion series and the observed intensities for the different charge states shown in A.

Fig. 5.

Histogram showing the relative populations of the different doubly stripped oligomers (n – 2) formed by αB-crystallin, calculated from intensities of the peaks in the MS/MS spectrum. The population of oligomers in the original sample (n) was calculated from the doubly stripped (n – 2) data.

Fig. 6.

Expansion of the spectrum containing the n – 2 oligomer series acquired at a voltage applied to the collision cell of 200 V (Fig. 2B) without selection of a precursor ion window. In this experiment the entire population of ions present in the sample was subjected to high-energy gas-phase collisions, giving rise to stripped oligomers for the full range of constituent assemblies. Nonoverlapping peaks proximal to the peak at 20,080 m/z provided additional information regarding the largest oligomers present in this sample.