The development of new ion-activation/dissociation methods continues to be one of the most active areas of mass spectrometry owing to the broad applications of tandem mass spectrometry in the ...identification and structural characterization of molecules. This Review will showcase the impact of ultraviolet photodissociation (UVPD) as a frontier strategy for generating informative fragmentation patterns of ions, especially for biological molecules whose complicated structures, subtle modifications, and large sizes often impede molecular characterization. UVPD energizes ions via absorption of high-energy photons, which allows access to new dissociation pathways relative to more conventional ion-activation methods. Applications of UVPD for the analysis of peptides, proteins, lipids, and other classes of biologically relevant molecules are emphasized in this Review.
Protein–protein interfaces and architecture are critical to the function of multiprotein complexes. Mass spectrometry-based techniques have emerged as powerful strategies for characterization of ...protein complexes, particularly for heterogeneous mixtures of structures. In the present study, activation and dissociation of three tetrameric protein complexes (streptavidin, transthyretin, and hemoglobin) in the gas phase was undertaken by 193 nm ultraviolet photodissociation (UVPD) for the characterization of higher order structure. High pulse energy UVPD resulted in the production of dimers and low charged monomers exhibiting symmetrical charge partitioning among the subunits (the so-called symmetrical dissociation pathways), consistent with the subunit organization of the complexes. In addition, UVPD promoted backbone cleavages of the monomeric subunits, the abundances of which corresponded to the more flexible loop regions of the proteins.
Characterization of all gas-phase charge sites of natively sprayed proteins and peptides is demonstrated using 193 nm UVPD. The high sequence coverage offered by UVPD is exploited for the accurate ...determination of charge sites in protein systems up to 18 kDa, allowing charge site to be studied as a function of protein conformation and the presence of disulfide bonds. Charging protons are found on both basic sidechains and on the amide backbone of less basic amino acids such as serine, glutamine, and proline. UVPD analysis was performed on the 3+ charge state of melittin, the 5+ to 8+ charge states of ubiquitin, and the 8+ charge state of reduced and oxidized β-lactoglobulin. The location of charges in gas-phase proteins is known to impact structure; molecular modeling of different charge site motifs of 3+ melittin demonstrates how placement of protons in simulations can dramatically impact the predicted structure of the molecule. The location of positive charge sites in ubiquitin and β-lactoglobulin are additionally found to depend on the presence or absence of salt-bridges, columbic repulsion across the length of the peptide, and protein conformation. Charge site isomers are demonstrated for ubiquitin and β-lactoglobulin but found to be much less numerous than previously predicted.
Patent foramen ovale (PFO) is prevalent in patients with migraine with aura. Observational studies show that PFO closure resulted in migraine cessation or improvement in approximately 80% of such ...patients. We investigated the effects of PFO closure for migraine in a randomized, double-blind, sham-controlled trial.
Patients who suffered from migraine with aura, experienced frequent migraine attacks, had previously failed > or = 2 classes of prophylactic treatments, and had moderate or large right-to-left shunts consistent with the presence of a PFO were randomized to transcatheter PFO closure with the STARFlex implant or to a sham procedure. Patients were followed up for 6 months. The primary efficacy end point was cessation of migraine headache 91 to 180 days after the procedure. In total, 163 of 432 patients (38%) had right-to-left shunts consistent with a moderate or large PFO. One hundred forty-seven patients were randomized. No significant difference was observed in the primary end point of migraine headache cessation between implant and sham groups (3 of 74 versus 3 of 73, respectively; P=0.51). Secondary end points also were not achieved. On exploratory analysis, excluding 2 outliers, the implant group demonstrated a greater reduction in total migraine headache days (P=0.027). As expected, the implant arm experienced more procedural serious adverse events. All events were transient.
This trial confirmed the high prevalence of right-to-left shunts in patients with migraine with aura. Although no significant effect was found for primary or secondary end points, the exploratory analysis supports further investigation. The robust design of this study has served as the model for larger trials that are currently underway in the United States and Europe.
Electron-based fragmentation methods have revolutionized biomolecular mass spectrometry, in particular native and top-down protein analysis. Here, we report the use of a new electromagnetostatic cell ...to perform electron capture dissociation (ECD) within a quadrupole/ion mobility/time-of-flight mass spectrometer. This cell was installed between the ion mobility and time-of-flight regions of the instrument, and fragmentation was fast enough to be compatible with mobility separation. The instrument was already fitted with electron transfer dissociation (ETD) between the quadrupole and mobility regions prior to modification. We show excellent fragmentation efficiency for denatured peptides and proteins without the need to trap ions in the gas phase. Additionally, we demonstrate native top-down backbone fragmentation of noncovalent protein complexes, leading to comparable sequence coverage to what was achieved using the instrument’s existing ETD capabilities. Limited collisional ion activation of the hemoglobin tetramer before ECD was reflected in the observed fragmentation pattern, and complementary ion mobility measurements prior to ECD provided orthogonal evidence of monomer unfolding within this complex. The approach demonstrated here provides a powerful platform for both top-down proteomics and mass spectrometry-based structural biology studies.
Determination of structure and folding of certain classes of proteins remains intractable by conventional structural characterization strategies and has spurred the development of alternative ...methodologies. Mass spectrometry-based approaches have a unique capacity to differentiate protein heterogeneity due to the ability to discriminate populations, whether minor or major, featuring modifications or complexation with non-covalent ligands on the basis of m/z. Cleavage of the peptide backbone can be further utilized to obtain residue-specific structural information. Here, hydrogen elimination monitoring (HEM) upon ultraviolet photodissociation (UVPD) of proteins transferred to the gas phase via nativespray ionization is introduced as an innovative approach to deduce backbone hydrogen bonding patterns. Using well-characterized peptides and a series of proteins, prediction of the engagement of the amide carbonyl oxygen of the protein backbone in hydrogen bonding using UVPD-HEM is demonstrated to show significant agreement with the hydrogen-bonding motifs derived from molecular dynamics simulations and X-ray crystal structures.
In solution, α-helices are stabilized at the termini by a variety of different capping interactions. Study of these interactions in the gas phase provides a unique means to explore the intrinsic ...properties that cause this stabilization. Evidence of helical and globular conformations is presented here for gas-phase, doubly charged peptides of sequence XA n K, wherein X is D, N, Q, or L. The relative abundance of the helical conformation is found to vary as a function of peptide length and the identity of the first amino acid, consistent with solution phase studies that have looked at the identity of the first amino acid. The N-terminal, b ion fragments of the doubly charged precursor peptides are shown to form helical and globular conformations. The stability of the helical fragments is examined as a function of fragment length, N-terminal amino acid, precursor conformation, and the activation energy used to generate the fragment. At lower collision energies, helical b ions preferentially form, particularly from helical precursors. The abundance of the helical b ion population is observed to dramatically decrease for NA n and DA n b ions smaller than the b10; simulations suggest this feature is due to the b10 having two complete turns of the helix, while the b9 and smaller ions have only a partial second turn, suggesting the b10 is the lower limit for stable helical conformations in b ions. Use of higher collision energies promotes the formation of globular structures in the b ions. This characteristic is attributed to increased conformational dynamics and subsequently improved proton transfer kinetics from the b ion’s C-terminal oxazolone ring to the N-terminus.
Covid-19 pandemic outbreak is the reason of the current world health crisis. The development of effective antiviral compounds and vaccines requires detailed descriptive studies of SARS-CoV-2 ...proteins. The SARS-CoV-2 spike (S) protein mediates virion binding to the human cells through its interaction with the ACE2 cell surface receptor and is one of the prime immunization targets. A functional virion is composed of three S1 and three S2 subunits created by furin cleavage of the spike protein at R682, a polybasic cleavage site that differs from the SARS-CoV spike protein of 2002. By analysis of the protein produced in HEK293 cells, we observe that the spike is O-glycosylated on a threonine (T678) near the furin cleavage site occupied by core-1 and core-2 structures. In addition, we have identified eight additional O-glycopeptides on the spike glycoprotein and confirmed that the spike protein is heavily N-glycosylated. Our recently developed liquid chromatography–mass spectrometry methodology allowed us to identify LacdiNAc structural motifs on all occupied N-glycopeptides and polyLacNAc structures on six glycopeptides of the spike protein. In conclusion, our study substantially expands the current knowledge of the spike protein’s glycosylation and enables the investigation of the influence of O-glycosylation on its proteolytic activation.
Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains that vary in their length and connectivity. Because of the challenging nature of characterizing these ...molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3′ position, followed by primary acyl chain fragmentation at both the 3′ and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages.
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