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1.
J Am Soc Mass Spectrom ; 35(7): 1377-1378, 2024 Jul 03.
Article in English | MEDLINE | ID: mdl-38957081
2.
J Am Soc Mass Spectrom ; 35(7): 1394-1402, 2024 Jul 03.
Article in English | MEDLINE | ID: mdl-38905538

ABSTRACT

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.


Subject(s)
Ion Mobility Spectrometry , Ion Mobility Spectrometry/methods , Tandem Mass Spectrometry/methods , Proteomics/methods , Pyruvate Kinase/chemistry , Pyruvate Kinase/analysis , Streptavidin/chemistry , Streptavidin/analysis , Cholera Toxin/analysis , Cholera Toxin/chemistry , Avidin/chemistry , Avidin/analysis , Proteins/analysis , Proteins/chemistry
3.
Chem Commun (Camb) ; 60(52): 6655-6658, 2024 Jun 25.
Article in English | MEDLINE | ID: mdl-38856910

ABSTRACT

We report the first mass photometric characterization of nanoaggregates of atomically precise nanoclusters (NCs) in solution. The differently-sized nanoaggregates of silver-gold alloy NCs, [Ag11-xAux(DPPB)5Cl5O2]2+ [x = 1-5 and DPPB = 1,4-bis(diphenylphosphino)butane], formed in solution, were examined by mass photometry (MP) with a protein calibration. In addition, we conducted MP studies of varying solvent composition to understand the structural evolution of nanoaggregates. The masses of nanoaggregates were correlated to structures of 15 to 50 nm diameter observed in cryo-electron microscopy.

4.
bioRxiv ; 2024 Apr 28.
Article in English | MEDLINE | ID: mdl-38712154

ABSTRACT

Native proteomics aims to measure endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separation techniques. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate), and those studies are either too time and labor-consuming or only able to detect small proteoforms or protein complexes. Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range Orbitrap mass spectrometer (UHMR). The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 100 pg of protein material, indicating the extremely high sensitivity of the technique. nCZE-MS analysis of an E . coli cell lysate detected 76 and 21 proteoforms or protein complexes in a mass range of 30-400 kDa and over 110 kDa, respectively, in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough of native proteomics for measuring complex proteomes, suggesting that nCZE-MS might be developed as a central technique for native proteomics.

5.
bioRxiv ; 2024 May 05.
Article in English | MEDLINE | ID: mdl-38746386

ABSTRACT

Homotropic cooperativity is widespread in biological regulation. The homo-oligomeric ring-shaped trp RNA binding attenuation protein (TRAP) from bacillus binds multiple tryptophan ligands (Trp) and becomes activated to bind a specific sequence in the 5' leader region of the trp operon mRNA. Ligand-activated binding to this specific RNA sequence regulates downstream biosynthesis of Trp in a feedback loop. Characterized TRAP variants form 11- or 12-mer rings and bind Trp at the interface between adjacent subunits. Various studies have shown that a pair of loops that gate each Trp binding site is flexible in the absence of the ligand and become ordered upon ligand binding. Thermodynamic measurements of Trp binding have revealed a range of cooperative behavior for different TRAP variants, even if the averaged apparent affinities for Trp have been found to be similar. Proximity between the ligand binding sites, and the ligand-coupled disorder-to-order transition has implicated nearest-neighbor interactions in cooperativity. To establish a solid basis for describing nearest-neighbor cooperativity we engineered dodecameric (12-mer) TRAP variants constructed with two subunits connected by a flexible linker (dTRAP). We mutated one of the protomers such that only every other site was competent for Trp binding. Thermodynamic and structural studies using native mass spectrometry, NMR spectroscopy, and cryo-EM provided unprecedented detail into the thermodynamic and structural basis for the observed ligand binding cooperativity. Such insights can be useful for understanding allosteric control networks and for the development of new ones with defined ligand sensitivity and regulatory control.

6.
Nat Struct Mol Biol ; 2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38627580

ABSTRACT

As one of the most prevalent anti-phage defense systems in prokaryotes, Gabija consists of a Gabija protein A (GajA) and a Gabija protein B (GajB). The assembly and function of the Gabija system remain unclear. Here we present cryo-EM structures of Bacillus cereus GajA and GajAB complex, revealing tetrameric and octameric assemblies, respectively. In the center of the complex, GajA assembles into a tetramer, which recruits two sets of GajB dimer at opposite sides of the complex, resulting in a 4:4 GajAB supramolecular complex for anti-phage defense. Further biochemical analysis showed that GajA alone is sufficient to cut double-stranded DNA and plasmid DNA, which can be inhibited by ATP. Unexpectedly, the GajAB displays enhanced activity for plasmid DNA, suggesting a role of substrate selection by GajB. Together, our study defines a framework for understanding anti-phage immune defense by the GajAB complex.

7.
Electrophoresis ; 2024 Apr 05.
Article in English | MEDLINE | ID: mdl-38577828

ABSTRACT

Capillary electrophoresis (CE) interfaced to mass spectrometry (MS) with electrospray ionization typically incorporates acidic additives or organic solvents to assist in ionization. Vibrating sharp-edge spray ionization (VSSI) is a voltage-free method to interface CE and MS that does not require these additives, making it appealing for protein analyses. CE-VSSI nanoflow sheath separations are performed with low ionic strength aqueous solutions in the sheath to reduce suppression. Serine is also included in the sheath to reduce analyte adduction. Proteins are detected in the 2.5-10 µM range, corresponding to an injected mass range of 0.1-1.2 ng. The anionic proteins ß-lactoglobulin and transferrin are resolved using an unmodified fused silica capillary because they do not exhibit nonspecific surface adsorption. Conversely, separations of cationic proteins cytochrome c, ribonuclease A, and α-chymotrypsinogen A in an unmodified capillary require acidic background electrolytes to overcome adsorption. Alternatively, a semipermanent coating comprised self-assembled lipids overcomes surface adsorption at a neutral pH. Separations with zwitterionic and hybrid cationic coatings are complete within 15 or 6 min, respectively. The dimeric form of triosephosphate isomerase was observed at a 60 µM, corresponding to a mass of 19 ng, by dropping the temperature of the MS inlet.

8.
J Mol Biol ; 436(11): 168590, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38663547

ABSTRACT

Redß is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ-exonuclease (λ-exo), Redß is part of a two-component DNA recombination system involved in multiple aspects of genome maintenance. The proteins have been exploited in powerful methods for bacterial genome engineering in which Redß can anneal an electroporated oligonucleotide to a complementary target site at the lagging strand of a replication fork. Successful annealing in vivo requires the interaction of Redß with E. coli single-stranded DNA-binding protein (SSB), which coats the ssDNA at the lagging strand to coordinate access of numerous replication proteins. Previous mutational analysis revealed that the interaction between Redß and SSB involves the C-terminal domain (CTD) of Redß and the C-terminal tail of SSB (SSB-Ct), the site for binding of numerous host proteins. Here, we have determined the x-ray crystal structure of Redß CTD in complex with a peptide corresponding to the last nine residues of SSB (MDFDDDIPF). Formation of the complex is predominantly mediated by hydrophobic interactions between two phenylalanine side chains of SSB (Phe-171 and Phe-177) and an apolar groove on the CTD, combined with electrostatic interactions between the C-terminal carboxylate of SSB and Lys-214 of the CTD. Mutation of any of these residues to alanine significantly disrupts the interaction of full-length Redß and SSB proteins. Structural knowledge of this interaction will help to expand the utility of Redß-mediated recombination to a wider range of bacterial hosts for applications in synthetic biology.


Subject(s)
Bacteriophage lambda , DNA, Single-Stranded , DNA-Binding Proteins , Escherichia coli Proteins , Escherichia coli , Viral Proteins , Bacteriophage lambda/genetics , Bacteriophage lambda/metabolism , Binding Sites , Crystallography, X-Ray , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Escherichia coli/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Models, Molecular , Protein Binding , Protein Conformation , Viral Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics
9.
bioRxiv ; 2024 Jun 28.
Article in English | MEDLINE | ID: mdl-38496594

ABSTRACT

We illustrate the utility of native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), for the characterization of protein complex topology and glycoprotein heterogeneity. ECCR efficiently reduces the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurements to greater than 100,000 m/z. For pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) reduces the charge states and yields more topologically informative fragmentation. This is the first demonstration that ECCR yields more native-like SID fragmentation. ECCR also significantly improved mass and glycan heterogeneity measurements of heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer. Protein glycosylation is important for structural and functional properties and plays essential roles in many biological processes. The immense heterogeneity in glycosylation sites and glycan structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Without ECCR, average mass determination of glycoprotein complexes is available only through charge detection mass spectrometry or mass photometry. With narrow m/z selection windows followed by ECCR, multiple glycoform m/z values are apparent, providing quick global glycoform profiling and providing a future path for glycan localization on individual intact glycoforms.

10.
bioRxiv ; 2024 Feb 04.
Article in English | MEDLINE | ID: mdl-38352409

ABSTRACT

With a rise in antibiotic resistance and chronic infection, the metabolic response of Salmonella enterica serovar Typhimurium to various dietary conditions over time remains an understudied avenue for novel, targeted therapeutics. Elucidating how enteric pathogens respond to dietary variation not only helps us decipher the metabolic strategies leveraged for expansion but also assists in proposing targets for therapeutic interventions. Here, we use a multi-omics approach to identify the metabolic response of Salmonella enterica serovar Typhimurium in mice on both a fibrous diet and high-fat diet over time. When comparing Salmonella gene expression between diets, we found a preferential use of respiratory electron acceptors consistent with increased inflammation of the high-fat diet mice. Looking at the high-fat diet over the course of infection, we noticed heterogeneity of samples based on Salmonella ribosomal activity, which separated into three infection phases: early, peak, and late. We identified key respiratory, carbon, and pathogenesis gene expression descriptive of each phase. Surprisingly, we identified genes associated with host-cell entry expressed throughout infection, suggesting sub-populations of Salmonella or stress-induced dysregulation. Collectively, these results highlight not only the sensitivity of Salmonella to its environment but also identify phase-specific genes that may be used as therapeutic targets to reduce infection. Importance: Identifying novel therapeutic strategies for Salmonella infection that occur in relevant diets and over time is needed with the rise of antibiotic resistance and global shifts towards Western diets that are high in fat and low in fiber. Mice on a high-fat diet are more inflamed compared to those on a fibrous diet, creating an environment that results in more favorable energy generation for Salmonella . Over time on a high-fat diet, we observed differential gene expression across infection phases. Together, these findings reveal the metabolic tuning of Salmonella to dietary and temporal perturbations. Research like this, exploring the dimensions of pathogen metabolic plasticity, can pave the way for rationally designed strategies to control disease.

11.
J Am Soc Mass Spectrom ; 35(3): 646-652, 2024 Mar 06.
Article in English | MEDLINE | ID: mdl-38303101

ABSTRACT

At the 33rd ASMS Sanibel Meeting, on Membrane Proteins and Their Complexes, a morning roundtable discussion was held discussing the current challenges facing the field of native mass spectrometry and approaches to expanding the field to nonexperts. This Commentary summarizes the discussion and current initiatives to address these challenges.


Subject(s)
Membrane Proteins , Mass Spectrometry/methods
12.
bioRxiv ; 2024 Jan 19.
Article in English | MEDLINE | ID: mdl-38293109

ABSTRACT

Salmonella enterica serovar Typhimurium is a pervasive enteric pathogen and an ongoing global threat to public health. Ecological studies in the Salmonella impacted gut remain underrepresented in the literature, discounting the microbiome mediated interactions that may inform Salmonella physiology during colonization and infection. To understand the microbial ecology of Salmonella remodeling of the gut microbiome, here we performed multi-omics approaches on fecal microbial communities from untreated and Salmonella -infected mice. Reconstructed genomes recruited metatranscriptomic and metabolomic data providing a strain-resolved view of the expressed metabolisms of the microbiome during Salmonella infection. This data informed possible Salmonella interactions with members of the gut microbiome that were previously uncharacterized. Salmonella- induced inflammation significantly reduced the diversity of transcriptionally active members in the gut microbiome, yet increased gene expression was detected for 7 members, with Luxibacter and Ligilactobacillus being the most active. Metatranscriptomic insights from Salmonella and other persistent taxa in the inflamed microbiome further expounded the necessity for oxidative tolerance mechanisms to endure the host inflammatory responses to infection. In the inflamed gut lactate was a key metabolite, with microbiota production and consumption reported amongst transcriptionally active members. We also showed that organic sulfur sources could be converted by gut microbiota to yield inorganic sulfur pools that become oxidized in the inflamed gut, resulting in thiosulfate and tetrathionate that supports Salmonella respiration. Advancement of pathobiome understanding beyond inferences from prior amplicon-based approaches can hold promise for infection mitigation, with the active community outlined here offering intriguing organismal and metabolic therapeutic targets.

13.
Nat Struct Mol Biol ; 31(3): 413-423, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38177683

ABSTRACT

Escherichia coli Septu system, an anti-phage defense system, comprises two components: PtuA and PtuB. PtuA contains an ATPase domain, while PtuB is predicted to function as a nuclease. Here we show that PtuA and PtuB form a stable complex with a 6:2 stoichiometry. Cryo-electron microscopy structure of PtuAB reveals a distinctive horseshoe-like configuration. PtuA adopts a hexameric arrangement, organized as an asymmetric trimer of dimers, contrasting the ring-like structure by other ATPases. Notably, the three pairs of PtuA dimers assume distinct conformations and fulfill unique roles in recruiting PtuB. Our functional assays have further illuminated the importance of the oligomeric assembly of PtuAB in anti-phage defense. Moreover, we have uncovered that ATP molecules can directly bind to PtuA and inhibit the activities of PtuAB. Together, the assembly and function of the Septu system shed light on understanding other ATPase-containing systems in bacterial immunity.


Subject(s)
Bacteriophages , Inflammasomes , Cryoelectron Microscopy , Bacteriophages/metabolism , Adenosine Triphosphatases/metabolism , Escherichia coli/metabolism
14.
J Am Soc Mass Spectrom ; 35(2): 214-223, 2024 Feb 07.
Article in English | MEDLINE | ID: mdl-38215279

ABSTRACT

The complexity of the lipidome has necessitated the development of novel analytical approaches for the identification and structural analysis of morphologically diverse classes of lipids. At this time, a variety of dissociation techniques have been utilized to probe lipid decomposition pathways in search of structurally diagnostic fragment ions. Here, we investigate the application of surface-induced dissociation (SID), a fragmentation technique that imparts energy to the target molecule via collision with a coated surface, for the fragmentation of seven lipids across four major lipid subclasses. We have developed a tuning methodology for guiding the efficient operation of a previously developed custom SID device for molecules as small as ca. 300 Da with ion mobility analysis of the fragmentation products. SID fragmentation of the various lipids analyzed was found to generate fragment ions similar to those observed in CID spectra, but fragment ion lab frame onset energies were lower in SID due to the higher energy deposition via a more massive target. For the largest lipid evaluated (cardiolipin 18:1), SID produced chain fragment ions, which yielded analytically useful information regarding the composition of the acyl tails. Ion mobility provided an orthogonal dimension of separation and aided in assigning product ions to their precursors. Overall, the combination of SID and IM-MS is another potential methodology in the analytical toolkit for lipid structural analysis.


Subject(s)
Ion Mobility Spectrometry , Lipids , Ions/chemistry , Mass Spectrometry/methods
15.
J Am Soc Mass Spectrom ; 34(10): 2065, 2023 Oct 04.
Article in English | MEDLINE | ID: mdl-37789802
16.
Anal Chem ; 95(37): 13889-13896, 2023 09 19.
Article in English | MEDLINE | ID: mdl-37672632

ABSTRACT

Charge detection mass spectrometry (CDMS) enables the direct mass measurement of heterogeneous samples on the megadalton scale, as the charge state for a single ion is determined simultaneously with the mass-to-charge ratio (m/z). Surface-induced dissociation (SID) is an effective activation method to dissociate non-intertwined, non-covalent protein complexes without extensive gas-phase restructuring, producing various subcomplexes reflective of the native protein topology. Here, we demonstrate that using CDMS after SID on an Orbitrap platform offers subunit connectivity, topology, proteoform information, and relative interfacial strengths of the intact macromolecular assemblies. SID dissects the capsids (∼3.7 MDa) of adeno-associated viruses (AAVs) into trimer-containing fragments (3mer, 6mer, 9mer, 15mer, etc.) that can be detected by the individual ion mass spectrometry (I2MS) approach on Orbitrap instruments. SID coupled to CDMS provides unique structural insights into heterogeneous assemblies that are not readily obtained by traditional MS measurements.


Subject(s)
Capsid , Dependovirus , Mass Spectrometry , Software
18.
J Am Soc Mass Spectrom ; 34(10): 2166-2175, 2023 Oct 04.
Article in English | MEDLINE | ID: mdl-37590530

ABSTRACT

Charge partitioning during the dissociation of protein complexes in the gas phase is influenced by many factors, such as interfacial interactions, protein flexibility, protein conformation, and dissociation methods. In the present work, two cysteine-containing homodimer proteins, ß-lactoglobulin and α-lactalbumin, with the disulfide bonds intact and reduced, were used to gain insight into the charge partitioning behaviors of collision-induced dissociation (CID) and surface-induced dissociation (SID) processes. For these proteins, we find that restructuring dominates with CID and dissociation with symmetric charge partitioning dominates with SID, regardless of whether intramolecular disulfide bonds are oxidized or reduced. CID of the charge-reduced dimeric protein complex leads to a precursor with a slightly smaller collision cross section (CCS), greater stability, and more symmetrically distributed charges than the significantly expanded form produced by CID of the higher charged dimer. Collision-induced unfolding plots demonstrate that the unfolding-restructuring of the protein complexes initiates the charge migration of higher charge-state precursors. Overall, gas collisions reveal the charge-dependent restructuring/unfolding properties of the protein precursor, while surface collisions lead predominantly to more charge-symmetric monomer separation. CID's multiple low-energy collisions sequentially reorganize intra- and intermolecular bonds, while SID's large-step energy jump cleaves intermolecular interfacial bonds in preference to reorganizing intramolecular bonds. The activated population of precursors that have taken on energy without dissociating (populated in CID over a wide range of collision energies, populated in SID for only a narrow distribution of collision energies near the onset of dissociation) is expected to be restructured, regardless of the activation method.

19.
bioRxiv ; 2023 Jun 30.
Article in English | MEDLINE | ID: mdl-37425951

ABSTRACT

Cellular production of tryptophan is metabolically expensive and tightly regulated. The small Bacillus subtilis zinc binding Anti-TRAP protein (AT), which is the product of the yczA/rtpA gene, is upregulated in response to accumulating levels of uncharged tRNATrp through a T-box antitermination mechanism. AT binds to the undecameric ring-shaped protein TRAP (trp RNA Binding Attenuation Protein), thereby preventing it from binding to the trp leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the trp operon. AT principally adopts two symmetric oligomeric states, a trimer (AT3) featuring a three-helix bundle, or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form has been shown to bind and inhibit TRAP. We demonstrate the utility of native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) for monitoring the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we report the use of solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT3, while heteronuclear 15N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT3 and binding-inactive AT12, with implications for TRAP inhibition.

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