Figure 1. Native mass spectrometry analysis of wt PCAT1 and PCAT1(C21A)-CtA complexes.

(A) wt PCAT1 and (B) PCAT1(C21A)-CtA complex. The samples were analyzed in 200 mM ammonium acetate containing either UDM or C₈E₄ at 2X critical micelle concentration (CMC). The peak series for the protein complex were at lower charge states in C₈E₄ (charged-reduced) than in UDM, consistent with previous native MS results of other membrane protein complexes (Reading et al., 2015). Note that the peak intensities for the 172 kDa complex in the deconvoluted spectra in (B) includes both the complex present in-solution and the subcomplex resulting from the gas-phase dissociation of the 182 kDa complex upon collision activation (for estimation of the % relative abundances of the complexes with correction for gas-phase dissociation, see Figure 1—figure supplement 2).

Figure 1—figure supplement 1. Native MS analysis of the wt PCAT1 at varying activation energies.

Native MS spectra for PCAT1 with CtA in UDM with variable (A) in-source dissociation (ISD) and (B) higher-energy collisional dissociation (HCD), and for C21A-PCAT1 in C₈E₄ with variable (C) HCD. The activation parameters were screened to determine the minimal collision activation energy required for detergent clean-up that leads to a well-resolved, near-baseline spectrum without dissociating the protein complex in the gas phase. Higher collision activation energies were needed for optimal native MS analysis of samples in UDM (ISD: 150–200 V and HCD: 150–200 V) than in C₈E₄ (ISD: 10 V and HCD: 150–200 V). Consequently, a peak series for the 81 kDa PCAT1 monomer can be observed at the lower m/z range for samples in UDM but not in C₈E₄.

Figure 1—figure supplement 2. Native MS analysis of the PCAT1(C21A) transporter in complex with CtA at varying activation energies.

Native MS spectra for PCAT1(C21A) with CtA in UDM with variable (A) ISD at constant HCD of 200 V, (B) HCD at constant ISD of 200 V, and (C) HCD at constant ISD of 10 V for samples in C₈E₄. The predominant peak series in all the spectra has a mass of 182 kDa (blue filled circles) corresponding to the PCAT(C21A) dimer + 2 CtA complex (1:2 complex). Note that most spectra had well-resolved, near-baseline spectra at various activation energy settings due to the removal of the detergent background. However, these parameters also led to gas-phase activation of the 182 kDa complex and subsequent ejection of one of the bound CtA yielding a charge-stripped subcomplex (172 kDa, PCAT(C21) dimer + 1 CtA complex). Hence, the charge-state series for the 172 kDa complex exhibits a bimodal distribution. The subgroup with higher charge states corresponds to a population that is present in-solution (hollow triangles) and another subgroup with lower charge states (filled triangles) corresponds to the population from gas-phase dissociation of the 182 kDa complex. To estimate the relative abundance of the 1:2 complex in the sample, the peak intensities for the 182 kDa complex and from the charge-stripped 172 kDa subcomplex (at z = 20+ to 24+ for UDM and z = 18+ to 22+ for C₈E₄) were combined and divided by the total peak intensities for all of the PCAT1 assemblies within each MS spectrum. The ionization and transmission efficiencies as well as MS response factors for the observed PCAT1 complexes might differ. Overall, the calculated relative abundance for the 182 kDa complex remained consistent for all samples within each detergent type at the activation energies that were tested. The lower abundance of the 182 kDa complex in UDM relative to C₈E₄ is mainly due to the higher collision activation energy settings required to remove the UDM detergent from the complex (i.e., 100–200 V ISD in UDM versus 10 V ISD in C₈E₄). When indicated, background subtraction was performed using the UniDec software with curved background and smoothing correction.