Desalting strategies for native mass spectrometry.

In native mass spectrometry (MS) salts are indispensable for preserving the native structures of biomolecules, but detrimental to mass sensitivity, resolution, and accuracy. Such a conflict makes desalting in native MS more challenging, distinctive, and sample-dependent than in peptide-centric MS. This review first briefly introduces the charged residue mechanism whereby native-like gaseous protein ions are released from electrospray droplets, revealing a higher degree of salt adduction than denatured proteins. Subsequently, this review summarizes and explores the existing strategies, underlying mechanisms and future perspectives of desalting in native MS. These strategies mainly focus on buffer exchange into volatile salts (offline and online approaches), addition of solution additives (e.g., anion, supercharging reagent, solution phase chelator and amino acid), use of submicron electrospray emitters (down to 60 nm), and other potential approaches (e.g., induced and electrophoretic nanoelectrospray ionization). The strategies of online buffer exchange and using nanoscale electrospray emitters are highlighted. This review would not only be a valuable addition to the field of sample preparation in MS, but would also serve as a beginner's guide to desalting in native MS.

A combination of multiple LC-MS approaches for the comprehensive characterization of cysteine-linked ADCs.

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.

Solution structure, dynamics and tetrahedral assembly of Anti-TRAP, a homo-trimeric triskelion-shaped regulator of tryptophan biosynthesis in Bacillus subtilis.

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 axially symmetric 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 three-fold axial symmetry or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form binds and inhibits TRAP. We apply native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) to monitor the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we use 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 binding and inhibition.

Detection of Noncovalent Protein-Ligand Complexes by IR-MALDESI-MS.

Native mass spectrometry (MS) is a powerful analytical technique to directly probe noncovalent protein-protein and protein-ligand interactions. However, not every MS platform can preserve proteins in their native conformation due to high energy deposition from the utilized ionization source. Most small molecules approved as drugs and in development interact with their targets through noncovalent interactions. Therefore, rapid methods to analyze noncovalent protein-ligand interactions are necessary for the early stages of the drug discovery pipeline. Herein, we describe a method for analyzing noncovalent protein-ligand complexes by IR-MALDESI-MS with analysis times of ∼13 s per sample. Carbonic anhydrase and the kinase domain of Bruton's tyrosine kinase are paired with known noncovalent binders to evaluate the effectiveness of native MS by IR-MALDESI.

Evaluation of a Commercial TIMS-Q-TOF Platform for Native Mass Spectrometry.

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.

Proteomic strategies to interrogate the Fe-S proteome.

Iron­sulfur (Fe-S) clusters, inorganic cofactors composed of iron and sulfide, participate in numerous essential redox, non-redox, structural, and regulatory biological processes within the cell. Though structurally and functionally diverse, the list of all proteins in an organism capable of binding one or more Fe-S clusters is referred to as its Fe-S proteome. Importantly, the Fe-S proteome is highly dynamic, with continuous cluster synthesis and delivery by complex Fe-S cluster biogenesis pathways. This cluster delivery is balanced out by processes that can result in loss of Fe-S cluster binding, such as redox state changes, iron availability, and oxygen sensitivity. Despite continued expansion of the Fe-S protein catalogue, it remains a challenge to reliably identify novel Fe-S proteins. As such, high-throughput techniques that can report on native Fe-S cluster binding are required to both identify new Fe-S proteins, as well as characterize the in vivo dynamics of Fe-S cluster binding. Due to the recent rapid growth in mass spectrometry, proteomics, and chemical biology, there has been a host of techniques developed that are applicable to the study of native Fe-S proteins. This review will detail both the current understanding of the Fe-S proteome and Fe-S cluster biology as well as describing state-of-the-art proteomic strategies for the study of Fe-S clusters within the context of a native proteome.

Biophysical insights into the dimer formation of human Sirtuin 2.

Sirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild-type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 (hSIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT hSIRT2 HDAC (residues 52-357) forms a homodimer in a concentration-dependent manner with a dimer-monomer dissociation constant of 8.3 ± 0.5 µM, which was determined by mass spectrometry. The dimer was disrupted into two monomers by binding to the HDAC inhibitors SirReal1 and SirReal2. We also confirmed dimer formation of hSIRT2 HDAC in living cells using a NanoLuc complementation reporter system. Examination of the relationship between dimer formation and deacetylation activity using several mutants of hSIRT2 HDAC revealed that some non-dimerizing mutants exhibited deacetylation activity for the N-terminal peptide of histone H3, similar to the wild type. The hSIRT2 HDAC mutant Δ292-306, which lacks a SIRT2-specific disordered loop region, was identified to exist as a monomer with slightly reduced deacetylation activity; the X-ray structure of the mutant Δ292-306 was almost identical to that of the WT hSIRT2 HDAC bound to an inhibitor. These results indicate that hSIRT2 HDAC forms a dimer, but this is independent of deacetylation activity. Herein, we discuss insights into the dimer formation of hSIRT2 based on our biophysical experimental results.

Epitope Mapping of Japanese Encephalitis Virus Neutralizing Antibodies by Native Mass Spectrometry and Hydrogen/Deuterium Exchange.

Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen-antibody and other protein-protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated.

Characterization of mAb size heterogeneity originating from a cysteine to tyrosine substitution using denaturing and native LC-MS.

Upon assessing the comparability between a biosimilar mAb and its reference product by non-reducing CE-SDS, increased levels of a heavy-heavy-light chain (HHL) variant, present as a low molecular weight (LMW) peak, were observed. RPLC-MS applied at top, middle-up and bottom-up level revealed the existence of Cys-to-Tyr substitutions, predominantly at position HC226 involved in connecting LC and HC, explaining the abundant HHL levels. Antigen binding was not impacted by the presence of this size variant suggesting a non-covalent association of Tyr substituted HHL and LC. The latter complex is not maintained in the denaturing conditions associated with CE-SDS and RPLC-MS. Its existence could, nevertheless, be confirmed by native SEC-MS which preserves non-covalent protein interactions during separation and electrospray ionization. Amino acid analysis furthermore demonstrated a depletion of Cys during the fed-batch process indicating that the observed size/sequence variant is not of genetic but rather of metabolic origin. Native SEC-MS showed that supplementing the cell culture medium with Cys halts misincorporation of Tyr and promotes the formation of the desired mAb structure. To the best of our knowledge, Cys-to-Tyr substitutions preventing interchain disulfide bridge formation have not been described earlier. This observation adds to the impressive structural heterogeneity reported to date for mAbs.

The solute carrier SPNS2 recruits PI(4,5)P2 to synergistically regulate transport of sphingosine-1-phosphate.

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.

How Do Cancer-Related Mutations Affect the Oligomerisation State of the p53 Tetramerisation Domain?

Tumour suppressor p53 plays a key role in the development of cancer and has therefore been widely studied in recent decades. While it is well known that p53 is biologically active as a tetramer, the tetramerisation mechanism is still not completely understood. p53 is mutated in nearly 50% of cancers, and mutations can alter the oligomeric state of the protein, having an impact on the biological function of the protein and on cell fate decisions. Here, we describe the effects of a number of representative cancer-related mutations on tetramerisation domain (TD) oligomerisation defining a peptide length that permits having a folded and structured domain, thus avoiding the effect of the flanking regions and the net charges at the N- and C-terminus. These peptides have been studied under different experimental conditions. We have applied a variety of techniques, including circular dichroism (CD), native mass spectrometry (MS) and high-field solution NMR. Native MS allows us to detect the native state of complexes maintaining the peptide complexes intact in the gas phase; the secondary and quaternary structures were analysed in solution by NMR, and the oligomeric forms were assigned by diffusion NMR experiments. A significant destabilising effect and a variable monomer population were observed for all the mutants studied.

A native multi-dimensional monitoring workflow for at-line characterization of mAb titer, size, charge, and glycoform heterogeneities in cell culture supernatant.

With growing maturity of the biopharmaceutical industry, new modalities entering the therapeutic design space and increasing complexity of formulations such as combination therapy, the demands and requirements on analytical workflows have also increased. A recent evolution in newer analytical workflows is that of multi-attribute monitoring workflows designed on chromatography-mass spectrometry (LC-MS) platform. In comparison to traditional one attribute per workflow paradigm, multi-attribute workflows are designed to monitor multiple critical quality attributes through a single workflow, thus reducing the overall time to information and increasing efficiency and throughput. While the 1st generation multi-attribute workflows focused on bottom-up characterization following peptide digestion, the more recent workflows have been focussing on characterization of intact biologics, preferably in native state. So far intact multi-attribute monitoring workflows suitable for comparability, utilizing single dimension chromatography coupled with MS have been published. In this study, we describe a native multi-dimensional multi-attribute monitoring workflow for at-line characterization of monoclonal antibody (mAb) titer, size, charge, and glycoform heterogeneities directly in cell culture supernatant. This has been achieved through coupling ProA in series with size exclusion chromatography in 1st dimension followed by cation exchange chromatography in the 2nd dimension. Intact paired glycoform characterization has been achieved through coupling 2D-LC with q-ToF-MS. The workflow with a single heart cut can be completed in 25 mins and utilizes 2D-liquid chromatography (2D-LC) to maximize separation and monitoring of titer, size as well as charge variants.

Coherent diffractive imaging of proteins and viral capsids: simulating MS SPIDOC.

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm-1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

Characterization of high-molecular weight by-products in the production of a trivalent bispecific 2+1 heterodimeric antibody.

The development of increasingly complex antibody formats, such as bispecifics, can lead to the formation of increasingly complex high- and low-molecular-weight by-products. Here, we focus on the characterization of high molecular weight species (HMWs) representing the highest complexity of size variants. Standard methods used for product release, such as size exclusion chromatography (SEC), can separate HMW by-products from the main product, but cannot distinguish smaller changes in mass. Here, for the identification of the diverse and complex HMW variants of a trivalent bispecific CrossMAb antibody, offline fractionation, as well as production of HMW by-products combined with comprehensive analytical testing, was applied. Furthermore, HMW variants were analyzed regarding their chemical binding nature and tested in functional assays regarding changes in potency of the variants. Changes in potency were explained by detailed characterization using mass photometry, SDS-PAGE analysis, native mass spectrometry (MS) coupled to SEC and bottom-up proteomics. We identified a major portion of the HMW by-products to be non-covalently linked, leading to dissociation and changes in activity. We also identified and localized high heterogeneity of a by-product of concern and applied a CD3 affinity column coupled to native MS to annotate unexpected by-products. We present here a multi-method approach for the characterization of complex HMW by-products. A better understanding of these by-products is beneficial to guide analytical method development and proper specification setting for therapeutic bispecific antibodies to ensure constant efficacy and patient safety of the product through the assessment of by-products.

Advances in high-resolution mass spectrometry techniques for analysis of high mass-to-charge ions.

A major challenge in modern mass spectrometry (MS) is achieving high mass resolving power and accuracy for precision analyses in high mass-to-charge (m/z) regions. To advance the capability of MS for increasingly demanding applications, understanding limitations of state-of-the-art techniques and their status in applied sciences is essential. This review summarizes important instruments in high-resolution mass spectrometry (HRMS) and related advances to extend their working range to high m/z regions. It starts with an overview of HRMS techniques that provide adequate performance for macromolecular analysis, including Fourier-transform, time-of-flight (TOF), quadrupole-TOF, and related data-processing techniques. Methodologies and applications of HRMS for characterizing macromolecules in biochemistry and material sciences are summarized, such as top-down proteomics, native MS, drug discovery, structural virology, and polymer analyses.

Mass spectrometry in gene therapy: Challenges and opportunities for AAV analysis.

The characterization of adeno-associated virus (AAV)-based gene therapy products represents significant challenges owing to their extremely large molecular sizes, structural complexity and heterogeneity, and limited sample amounts. Mass spectrometry (MS) is one of the key analytical tools that can overcome these challenges and serve as an important technique for the analysis of multiple attributes. In this review, the current methodologies and emerging trends in MS analysis of AAV gene therapy products are presented, highlighting their advantages and unique capabilities in addressing key issues encountered in intact AAV vector analysis, capsid viral protein characterization and impurity analysis.

The multifaceted roles of mass spectrometric analysis in nucleic acids drug discovery and development.

The deceptively simple concepts of mass determination and fragment analysis are the basis for the application of mass spectrometry (MS) to a boundless range of analytes, including fundamental components and polymeric forms of nucleic acids (NAs). This platform affords the intrinsic ability to observe first-hand the effects of NA-active drugs on the chemical structure, composition, and conformation of their targets, which might affect their ability to interact with cognate NAs, proteins, and other biomolecules present in a natural environment. The possibility of interfacing with high-performance separation techniques represents a multiplying factor that extends these capabilities to cover complex sample mixtures obtained from organisms that were exposed to NA-active drugs. This report provides a brief overview of these capabilities in the context of the analysis of the products of NA-drug activity and NA therapeutics. The selected examples offer proof-of-principle of the applicability of this platform to all phases of the journey undertaken by any successful NA drug from laboratory to bedside, and provide the rationale for its rapid expansion outside traditional laboratory settings in support to ever growing manufacturing operations.

Determination of label efficiency and label degree of critical reagents by LC-MS and native MS.

Degree of labeling and label efficiency are key factors for optimal characterization of critical reagents that are used in ligand binding assays. Here, three case studies are shown demonstrating how liquid chromatography-mass spectrometry (LC-MS) was utilized to characterize critical reagents using three unique methodologies. Critical reagent batches were prepared for LC-MS analysis by use of: 20 mM dithiothreitol (DTT) (Case 1), rapid PNGaseF (Case 2), and a mobile phase diluent (Case 3). LC-MS was run at three different MS method conditions in each troubleshooting case specific for reduced IgG, intact IgG, and native LC-MS, respectively. Specified LC-MS methods based on sample type and configuration elucidated clear MS profiles, allowing for degree of labeling and label efficiencies to be calculated. Ultimately the LC-MS analyses were fine-tuned for critical reagent characterization, and practices for analyzing similar reagents in the future can be established.

Cryo-EM structure of the diapause chaperone artemin.

The protein artemin acts as both an RNA and protein chaperone and constitutes over 10% of all protein in Artemia cysts during diapause. However, its mechanistic details remain elusive since no high-resolution structure of artemin exists. Here we report the full-length structure of artemin at 2.04 Å resolution. The cryo-EM map contains density for an intramolecular disulfide bond between Cys22-Cys61 and resolves the entire C-terminus extending into the core of the assembled protein cage but in a different configuration than previously hypothesized with molecular modeling. We also provide data supporting the role of C-terminal helix F towards stabilizing the dimer form that is believed to be important for its chaperoning activity. We were able to destabilize this effect by placing a tag at the C-terminus to fully pack the internal cavity and cause limited steric hindrance.

Microflow size exclusion chromatography to preserve micromolar affinity complexes and achieve subunit separations for native state mass spectrometry.

For high throughput native mass spectrometry (MS) protein characterization, it is advantageous to desalt and separate proteins by size exclusion chromatography (SEC). Sensitivity, resolution, and speed in these methods remain limited by standard SEC columns. Moreover, the efficient packing of small bore columns is notoriously difficult. SEC sensitivity is inherently limited because solutes are not focused into concentrated bands and low affinity native complexes may dissociate on column. Recent work evaluated the suitability of crosslinked gel media in small bore formats for online desalting. Here, small bore format online SEC for native MS studies is again investigated but with alternative materials. We systematically studied the utility of diol and hydroxy terminated polyethylene oxide (PEO) bonded 1.7 µm organosilica particles as packed into 1 mm ID stainless steel (SS) hardware and hardware treated with hydrophilic hybrid surface technology (h-HST). For the equivalent diol-bonded particle and hardware, UV limits of detection (LODs) were reduced 32 to 89% with a microflow separation (15 µL/min) on a 1 × 50 mm column as compared to a 4.6 × 150 mm high-flow separation (300 µL/min) at the same linear velocity. Run times were also shortened by 45%. A switch from SS to h-HST hardware led to a significant reduction in secondary interactions and a corresponding improvement in detection limits for trastuzumab, myoglobin, IgG and albumin for both UV and MS. Coupling of the small bore columns to multichannel microflow emitters resulted in 10 to 100-fold gains in MS sensitivity, depending on the analyte. MS LOD values were significantly reduced into the low attomole ranges. Columns were then evaluated for their effects on the preservation of complexes, including concanavalin A, in its apo and ligand-bound states, and three therapeutically relevant noncovalent systems previously undetected on large column formats. The results suggest that the detection of large complexes by SEC is not just a function of sensitivity but is directly affected by chemical secondary interactions. The ability to detect 0.1 to 1 MDa complexes, with between 1 and 40 micromolar dissociation constants, represents a critical advancement for high-throughput native MS workflows as applied to the analysis of therapeutics.