Dissecting the Heterogeneous Glycan Profiles of Recombinant Coronavirus Spike Proteins with Individual Ion Mass Spectrometry.

Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2, play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we utilized individual ion mass spectrometry (I2MS), a multiplexed charge detection measurement with similarities to charge detection mass spectrometry (CDMS), in which a commercially available Orbitrap analyzer is used to directly produce mass profiles of these heterogeneous coronavirus spike protein trimers under native-like conditions. Analysis by I2MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques, highlighting the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I2MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N-glycans followed by I2MS analysis indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts, and these differences are correlated with solvent accessibility. I2MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional mass analysis methods such as size exclusion chromatography-multiangle light scattering (SEC-MALS) and field flow fractionation-multiangle light scattering (FFF-MALS). An added advantage of I2MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I2MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development.

De novo design of transmembrane ß barrels.

Transmembrane ß-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes and form stable pores, but the range of pore properties that can be achieved by repurposing natural TMBs is limited. We leverage the power of de novo computational design coupled with a "hypothesis, design, and test" approach to determine TMB design principles, notably, the importance of negative design to slow ß-sheet assembly. We design new eight-stranded TMBs, with no homology to known TMBs, that insert and fold reversibly into synthetic lipid membranes and have nuclear magnetic resonance and x-ray crystal structures very similar to the computational models. These advances should enable the custom design of pores for a wide range of applications.

Coupling 193 nm Ultraviolet Photodissociation and Ion Mobility for Sequence Characterization of Conformationally-Selected Peptides.

Ultraviolet photodissociation (UVPD) has emerged as a useful technique for characterizing peptide, protein, and protein complex primary and secondary structure. 193 nm UVPD, specifically, enables extensive covalent fragmentation of the peptide backbone without the requirement of a specific side chain chromophore and with no precursor charge state dependence. We have modified a commercial quadrupole-ion mobility-time-of-flight (Q-IM-TOF) mass spectrometer to include 193 nm UVPD following ion mobility. Ion mobility (IM) is a gas-phase separation technique that enables separation of ions by their size, shape, and charge, providing an orthogonal dimension of separation to mass analysis. Following instrument modifications, we characterized the performance of, and information that could be generated from, this new setup using the model peptides substance P, melittin, and insulin chain B. These experiments show extensive fragmentation across the peptide backbone and a variety of ion types as expected from 193 nm UVPD. Additionally, y-2 ions (along with complementary a+2 and b+2 ions) N-terminal to proline were observed. Combining the IM separation and mobility gating capabilities with UVPD, we demonstrate the ability to accomplish both mass- and mobility-selection of bradykinin des-Arg9 and des-Arg1 peptides followed by complete sequence characterization by UVPD. The new capabilities of this modified instrument demonstrate the utility of combining IM with UVPD because isobaric species cannot be independently selected with a traditional quadrupole alone.

Collision Cross Sections of Charge-Reduced Proteins and Protein Complexes: A Database for Collision Cross Section Calibration.

The use of charge-reducing reagents to generate lower-charge ions has gained popularity in the field of native mass spectrometry (MS) and ion mobility mass spectrometry (IM-MS). This is because the lower number of charged sites decreases the propensity for Coulombic repulsions and unfolding/restructuring, helping to preserve the native-like structure. Furthermore, lowering the charge state consequently increases the mass-to-charge values (m/z), effectively increasing spacing between signals originating from small mass differences, such as different proteoforms or protein-drug complexes. IM-MS yields collision cross section (CCS, Ω) values that provide information about the three-dimensional structure of the ion. Traveling wave IM (TWIM) is an established and expanding technique within the native MS field. TWIM measurements require CCS calibration, which is achieved via the use of standard species of known CCS. Current databases for native-like proteins and protein complexes provide CCS values obtained using normal (i.e., non-charge-reducing) conditions. Herein, we explored the validity of using "normal" charge calibrants to calibrate for charge-reduced proteins and show cases where it is not appropriate. Using a custom linear field drift cell that enables the determination of ion mobilities from "first principles", we directly determined CCS values for 19 protein calibrant species under three solution conditions (yielding a broad range of charge states) and two drift gases. This has established a database of CCS and reduced-mobility (K0) values, along with their associated uncertainties, for proteins and protein complexes over a large m/z range. TWIM validation of this database shows improved accuracy over existing methods in calibrating CCS values for charge-reduced proteins.

A Tilted Surface and Ion Carpet Array for SID.

The development of native mass spectrometry (MS) has provided structural biologists an additional tool to probe the structures of large macromolecular systems. Surface-induced dissociation (SID) is one activation method used within tandem MS experiments that has proven useful in interrogating the connectivity and topology of biologically-relevant protein complexes. We present here the use of a tilted surface and ion carpet array within a new SID device design, enabling decreased dimensions along the ion path and fewer lenses to tune. This device works well in fragmenting ions of both low (peptides) and high (protein complexes) m/z. Results show that the ion carpet array, while enabling simplification of the back-end of the device, has deficiencies in product collection and subsequently signal at higher SID energies when fragmenting protein complexes. However, the use of the tilted surface is advantageous as an effective way to shorten the device and reduce the number of independent voltages.

Ion Mobility and Surface Collisions: Submicrometer Capillaries Can Produce Native-like Protein Complexes.

The use of submicrometer capillaries for nanoelectrospray ionization of native proteins and protein complexes effectively reduces the number of nonspecific salt adducts to biological molecules, therefore increasing the apparent resolution of a mass spectrometer without any further instrument modifications or increased ion activation. However, the increased interaction between proteins and the surface of the capillary has been shown to promote protein expansion and therefore loss of native structure. Here, we compare the effect of micrometer and submicrometer sized capillaries on the native structures of the protein complexes streptavidin, concanavalin A, and C-reactive protein under charge reducing conditions. We observe that the use of submicrometer capillaries did not result in a significantly higher charge state distribution, indicative of expansion, when compared to micrometer sized capillaries for complexes in 100 mM ammonium acetate and 100 mM triethylammonium acetate and for streptavidin in 200 mM ammonium acetate with no charge reduction. Additionally, no significant differences in collision cross sections were observed using ion mobility mass spectrometry. Finally, the dissociation behaviors of protein complexes ionized using micrometer and submicrometer capillaries were compared to determine if any structural perturbation occurred during ionization. Protein complexes from both capillary sizes displayed similar surface-induced dissociation patterns at similar activation energies. The results suggest that submicrometer capillaries do not result in significant changes to protein complex structure under charge reducing conditions and may be used for native mass spectrometry experiments. Submicrometer capillaries can be used to resolve small mass differences of biological systems on a QTOF platform; however, a laser tip puller is required for pulling reproducible submicrometer capillaries, and disruption in spray due to clogging was observed for larger protein complexes.

Design and Performance of a Second-Generation Surface-Induced Dissociation Cell for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of Native Protein Complexes.

A second-generation ("Gen 2") device capable of surface-induced dissociation (SID) and collision-induced dissociation (CID) for Fourier transform ion cyclotron resonance mass spectrometry of protein complexes has been designed, simulated, fabricated, and experimentally compared to a first-generation device ("Gen 1"). The primary goals of the redesign were to (1) simplify SID by reducing the number of electrodes, (2) increase CID and SID sensitivity by lengthening the collision cell, and (3) increase the mass range of the device for analysis of larger multimeric proteins, all while maintaining the normal instrument configuration and operation. Compared to Gen 1, Gen 2 exhibits an approximately 10× increase in sensitivity in flythrough mode, 7× increase in CID sensitivity for protonated leucine enkephalin (m/z 556), and 14× increase of CID sensitivity of 53 kDa streptavidin tetramer. It also approximately doubles the useful mass range (from m/z 8000 to m/z 15 000) using a rectilinear ion trap with a smaller inscribed radius or triples it (to m/z 22 000) using a hexapole collision cell and yields a 3-10× increase in SID sensitivity. We demonstrate the increased mass range and sensitivity on a variety of model molecules spanning nearly 3 orders of magnitude in absolute mass and present examples where the high resolution of the FT-ICR is advantageous for deconvoluting overlapping SID fragments.