Mass spectrometry-based methods to study protein architecture and dynamics

Abstract

Mass spectrometry is now an indispensable tool in the armamentarium of molecular biophysics, where it is used for tasks ranging from protein sequencing and mapping of post-translational modifications to studies of higher order structure, conformational dynamics, and interactions of proteins with small molecule ligands and other biopolymers. This mini-review highlights several popular mass spectrometry-based tools that are now commonly used for structural studies of proteins beyond their covalent structure with a particular emphasis on hydrogen exchange and direct electrospray ionization mass spectrometry.

Figure 1

ESI mass spectra of human ferritin (light chain) acquired under the conditions described below. A: Solution (10 μM) in water/methanol/acetic acid, 48:50:2 (v:v:v) and minimal collisional activation in the ESI MS interface. B: Same solution conditions as above and significant collisional activation of the protein ions in the ESI MS interface. C: Solution (10 μM) in 20 mM aqueous ammonium acetate and minimal collisional activation in the ESI MS interface. Fragment ion labeling in panel B uses Biemann's nomenclature of polypeptide ion fragmentation.

Figure 2

ESI mass spectra of 0.9 mg/mL rhASA in 100 mM ammonium acetate at pH 7.5 and 5.0. The inset shows relative abundance of ionic signals corresponding to dimeric, tetrameric, and octameric species as a function of pH. Adapted with permission from Abzalimov et al., Anal Chem, 2013, 85, 1591–1596. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.] [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

ESI mass spectrum of GroEL acquired under near-native conditions in solution (100 mM ammonium acetate) and mild collisional activation in the ESI interface. The low m/z region of the spectrum is shown in the inset. Highly charged monomers and low charge density tridecamers are products of dissociation of tetradecameric structures in the gas phase. Oligomerization of GroEL tetradecamers (formation of 2M₁₄ species) is likely caused by increased protein concentration in ESI droplets as a result of solvent evaporation. Adapted with permission from Kaltashov and Abzalimov, J Am Soc Mass Spectrom, 2008, 19, 1239–1246.

Figure 4

ESI mass spectra of porcine pepsin acquired under near-native (pH 1.6, black trace) and denaturing (pH adjusted to 9.5, 50% MeOH by volume, blue and red traces) conditions. The mass spectra are acquired in the positive (black and blue traces) and negative (red) ion modes. The inset shows the crystal structure of pepsin, where acidic and basic residues are colored in red and blue, respectively. Adapted with permission from Kaltashov and Abzalimov, J Am Soc Mass Spectrom, 2008, 19, 1239–1246. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.] [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

Chemometric analysis of the protein ion charge state distributions in ESI mass spectra of aMb acquired over a wide range of near-native and nondenaturing conditions (acid unfolding). A: Plots of singular values w_i (the inset in this panel shows cumulative variance accounted for by the first i singular values as a function of i). B and C: Normalized projection coefficients p_ik, P = V^T obtained from SVD of the ESI MS data array (projection coefficients corresponding to only two representative significant [i = 1 and 4] and two insignificant [i = 6 and 8] singular values are shown for clarity). D–F: Fitting for representative charge state distributions of aMb, the spectra were acquired in 10 mM CH₃CO₂NH₄ aqueous solutions whose pH levels were adjusted to 7.4 (D), 4.5 (E), and 2.5 (F). G: Quality of the aMb ion signal reconstruction using first M singular values as a function of the dataset size K. The curves represent cumulative variance accounted for by the first M singular values within K ESI spectra of aMb (M = 2–8).

Figure 6

Time evolution of the ion peak (charge state +14) of cellular retinoic acid binding protein I in H₂O solution at pH 3.5 (the protein was completely deuterated and kept in D₂O-based solution prior to the measurement, and the HDX reactions were induced by rapid dilution of the stock solution in H₂O). The dashed line on the left indicates the position of the fully exchanged protein ion peak. Reproduced with permission from Eyles et al., J Mass Spectrom, 1999, 34, 1289–1295.

Figure 7

Schematic representation of HDX MS work flow to examine protein higher order structure and conformational dynamics. The exchange is initiated by placing the unlabeled protein into a D₂O-based solvent system (e.g., by a rapid dilution). Unstructured and highly dynamic protein segments undergo fast exchange (blue and red colors represent protons and deuterons, respectively). Following the quench step (rapid solution acidification and temperature drop), the protein loses its native conformation, but the spatial distribution of backbone amide protons and deuterons across the backbone is preserved (all labile hydrogen atoms at side chains undergo fast back-exchange at this step). Rapid clean-up followed by MS measurement of the protein mass reports the total number of backbone amide hydrogen atoms exchanged under native conditions (a global measure of the protein stability under native conditions), as long as the quench conditions are maintained during the sample work-up and measurement. Alternatively, the protein can by digested under the quench conditions using acid-stable protease(s), and LC/MS analysis of masses of individual proteolytic fragments will provide information on the backbone protection of corresponding protein segments under the native conditions. Reproduced with permission from Bobst and Kaltashov, Curr Pharm Biotechnol, 2011, 12, 1517–1529. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.] [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 8

Local HDX MS data for the N-lobe of human serum transferrin (a polypeptide segment represented by a peptic fragment 205–211). The top diagrams show localization of this segment within open (left) and closed (right) conformation of the protein (apo- and holo-forms, respectively). The mass spectra in the lower left panel show evolution of the isotopic distribution of this peptide throughout the course of exchange reactions at neutral pH. The red and blue traces correspond to peptides derived from holo- and apo-forms of the protein, respectively. The diagram in the lower right panel shows kinetics of deuterium incorporation within this peptide at neutral pH. Adapted with permission from Kaltashov et al., Biochim Biophys Acta, 2012, 1820, 417–426. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.] [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]