High-resolution Native Mass Spectrometry Research Articles

Deciphering Pathways and Thermodynamics of Protein Assembly Using Native Mass Spectrometry.

Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation. In contrast, high-resolution native mass spectrometry (nMS) can directly determine the stoichiometry of biomolecular complexes in vitro. However, quantification is hindered by the fact that the relative abundances of gas-phase ions generally do not reflect solution concentrations due to nonuniform response factors. Recently, slow mixing mode (SLOMO)-nMS, which can quantify the relative response factors of interacting species, has been demonstrated to reliably measure the affinity (Kd) of binary biomolecular complexes. Here, we introduce an extended form of SLOMO-nMS that enables simultaneous quantification of the thermodynamics in multistep association reactions. Application of this method to homo-oligomerization of concanavalin A and insulin confirmed the reliability of the assay and uncovered details about the assembly processes that had previously resisted elucidation. Results acquired using SLOMO-nMS implemented with charge detection shed new light on the binding of recombinant human angiotensin-converting enzyme 2 and the SARS-CoV-2 spike protein. Importantly, new assembly pathways were uncovered, and the affinities of these interactions, which regulate host cell infection, were quantified. Together, these findings highlight the tremendous potential of SLOMO-nMS to accelerate the characterization of protein assembly pathways and thermodynamics and, in so doing, enhance fundamental biological understanding and facilitate therapeutic development. https://orcid.org/0000-0002-3389-7112.

Water Plays Key Roles in Stabilities of Wild Type and Mutant Transthyretin Complexes.

Transthyretin (TTR), a 56 kDa homotetramer that is involved in the transport of thyroxine and retinol, has been linked to amyloidosis through disassembly of tetramers to form monomers, dimers, and trimers that then reassemble into higher order oligomers and/or fibrils. Hybrid TTR (hTTR) tetramers are found in heterozygous individuals that express both wild type TTR (wt-TTR) and mutant TTR (mTTR) forms of the protein, and these states display increased rates of amyloidosis. Here we monitor subunit exchange (SUE) reactions involving homomeric and mixed tetramers using high resolution native mass spectrometry (nMS). Our results show evidence that differences in TTR primary structure alter tetramer stabilities, and hTTR products can form spontaneously by SUE reactions. In addition, we find that solution temperature has strong effects on TTR tetramer stabilities and formation of SUE products. Lower temperatures promote formation of hTTR tetramers containing L55P and V30M subunits, whereas small effects on the formation of hTTR tetramers containing F87A and T119M subunits are observed. We hypothesize that the observed temperature dependent stabilities and subsequent SUE behavior are a result of perturbations to the network of "two kinds of water": hydrating and structure stabilizing water molecules (Spyrakis et al. J. Med. Chem. 2017, 60 (16), 6781-6827; Xu et al. Soft Matter 2012, 8, 324-336) that stabilize wt-TTR and mTTR tetramers. The results presented in this work illustrate the utility of high resolution nMS for studies of the structures, stabilities, and dynamics of protein complexes that directly influence SUE reactions.

Native ion mobility-mass spectrometry reveals the binding mechanisms of anti-amyloid therapeutic antibodies.

One of the most important attributes of anti-amyloid antibodies is their selective binding to oligomeric and amyloid aggregates. However, current methods of examining the binding specificities of anti-amyloid β (Aβ) antibodies have limited ability to differentiate between complexes that form between antibodies and monomeric or oligomeric Aβ species during the dynamic Aβ aggregation process. Here, we present a high-resolution native ion-mobility mass spectrometry (nIM-MS) method to investigate complexes formed between a variety of Aβ oligomers and three Aβ-specific IgGs, namely two antibodies with relatively high conformational specificity (aducanumab and A34) and one antibody with low conformational specificity (crenezumab). We found that crenezumab primarily binds Aβ monomers, while aducanumab preferentially binds Aβ monomers and dimers and A34 preferentially binds Aβ dimers, trimers, and tetrameters. Through collision induced unfolding (CIU) analysis, our data indicate that antibody stability is increased upon Aβ binding and, surprisingly, this stabilization involves the Fc region. Together, we conclude that nIM-MS and CIU enable the identification of Aβ antibody binding stoichiometries and provide important details regarding antibody binding mechanisms.

Proteoform-Resolved Profiling of Plasminogen Activation Reveals Novel Abundant Phosphorylation Site and Primary N-Terminal Cleavage Site

Plasminogen (Plg), the zymogen of plasmin (Plm), is a glycoprotein involved in fibrinolysis and a wide variety of other physiological processes. Plg dysregulation has been implicated in a range of diseases. Classically, human Plg is categorized into two types, supposedly having different functional features, based on the presence (type I) or absence (type II) of a single N-linked glycan. Using high-resolution native mass spectrometry, we uncovered that the proteoform profiles of human Plg (and Plm) are substantially more extensive than this simple binary classification. In samples derived from human plasma, we identified up to 14 distinct proteoforms of Plg, including a novel highly stoichiometric phosphorylation site at Ser339. To elucidate the potential functional effects of these post-translational modifications, we performed proteoform-resolved kinetic analyses of the Plg-to-Plm conversion using several canonical activators. This conversion is thought to involve at least two independent cleavage events: one to remove the N-terminal peptide and another to release the active catalytic site. Our analyses reveal that these processes are not independent but are instead tightly regulated and occur in a step-wise manner. Notably, N-terminal cleavage at the canonical site (Lys77) does not occur directly from intact Plg. Instead, an activation intermediate corresponding to cleavage at Arg68 is initially produced, which only then is further processed to the canonical Lys77 product. Based on our results, we propose a refined categorization for human Plg proteoforms. In addition, we reveal that the proteoform profile of human Plg is more extensive than that of rat Plg, which lacks, for instance, the here-described phosphorylation at Ser339.

Uncovering the Role of N-Glycan Occupancy on the Cooperative Assembly of Spike and Angiotensin Converting Enzyme 2 Complexes: Insights from Glycoengineering and Native Mass Spectrometry.

Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of N-glycan occupancy on the assembly of multiple Spike-ACE2 complexes. We confirmed that intact Spike trimers have all 66 N-linked sites occupied. For monomeric ACE2, all seven N-linked glycan sites are occupied to various degrees; six sites have >90% occupancy, while the seventh site (Asn690) is only partially occupied (∼30%). By resolving the glycoforms on ACE2, we deciphered the influence of each N-glycan on ACE2 dimerization. Unexpectedly, we found that Asn432 plays a role in mediating dimerization, a result confirmed by site-directed mutagenesis. We also found that glycosylated dimeric ACE2 and Spike trimers form complexes with multiple stoichiometries (Spike-ACE2 and Spike2-ACE2) with dissociation constants (Kds) of ∼500 and <100 nM, respectively. Comparing these values indicates that positive cooperativity may drive ACE2 dimers to complex with multiple Spike trimers. Overall, our results show that occupancy has a key regulatory role in mediating interactions between ACE2 dimers and Spike trimers. More generally, since soluble ACE2 (sACE2) retains an intact SARS-CoV-2 interaction site, the importance of glycosylation in ACE2 dimerization and the propensity for Spike and ACE2 to assemble into higher oligomers are molecular details important for developing strategies for neutralizing the virus.

Stochastic assembly of biomacromolecular complexes: impact and implications on charge interpretation in native mass spectrometry.

Native mass spectrometry is a potent method for characterizing biomacromolecular assemblies. A critical aspect to extracting accurate mass information is the correct inference of the ion ensemble charge states. While a variety of experimental strategies and algorithms have been developed to facilitate this, virtually all approaches rely on the implicit assumption that any peaks in a native mass spectrum can be directly attributed to an underlying charge state distribution. Here, we demonstrate that this paradigm breaks down for several types of macromolecular protein complexes due to the intrinsic heterogeneity induced by the stochastic nature of their assembly. Utilizing several protein assemblies of adeno-associated virus capsids and ferritin, we demonstrate that these particles can produce a variety of unexpected spectral appearances, some of which appear superficially similar to a resolved charge state distribution. When interpreted using conventional charge inference strategies, these distorted spectra can lead to substantial errors in the calculated mass (up to ∼5%). We provide a novel analytical framework to interpret and extract mass information from these spectra by combining high-resolution native mass spectrometry, single particle Orbitrap-based charge detection mass spectrometry, and sophisticated spectral simulations based on a stochastic assembly model. We uncover that these mass spectra are extremely sensitive to not only mass heterogeneity within the subunits, but also to the magnitude and width of their charge state distributions. As we postulate that many protein complexes assemble stochastically, this framework provides a generalizable solution, further extending the usability of native mass spectrometry in the characterization of biomacromolecular assemblies.

Charting the Proteoform Landscape of Serum Proteins in Individual Donors by High-Resolution Native Mass Spectrometry.

Most proteins in serum are glycosylated, with several annotated as biomarkers and thus diagnostically important and of interest for their role in disease. Most methods for analyzing serum glycoproteins employ either glycan release or glycopeptide centric mass spectrometry-based approaches, which provide excellent tools for analyzing known glycans but neglect previously undefined or unknown glycosylation and/or other co-occurring modifications. High-resolution native mass spectrometry is a relatively new technique for the analysis of intact glycoproteins, providing a “what you see is what you get” mass profile of a protein, allowing the qualitative and quantitative observation of all modifications present. So far, a disadvantage of this approach has been that it centers mostly on just one specific serum glycoprotein at the time. To address this issue, we introduce an ion-exchange chromatography-based fractionation method capable of isolating and analyzing, in parallel, over 20 serum (glyco)proteins, covering a mass range between 30 and 190 kDa, from 150 μL of serum. Although generating data in parallel for all these 20 proteins, we focus the discussion on the very complex proteoform profiles of four selected proteins, i.e., α-1-antitrypsin, ceruloplasmin, hemopexin, and complement protein C3. Our analyses provide an insight into the extensive proteoform landscape of serum proteins in individual donors, caused by the occurrence of various N- and O-glycans, protein cysteinylation, and co-occurring genetic variants. Moreover, native mass intact mass profiling also provided an edge over alternative approaches revealing the presence of apo- and holo-forms of ceruloplasmin and the endogenous proteolytic processing in plasma of among others complement protein C3. We also applied our approach to a small cohort of serum samples from healthy and diseased individuals. In these, we qualitatively and quantitatively monitored the changes in proteoform profiles of ceruloplasmin and revealed a substantial increase in fucosylation and glycan occupancy in patients with late-stage hepatocellular carcinoma and pancreatic cancer as compared to healthy donor samples.

Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent responses

Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3β or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies.

Cupric Ions Selectively Modulate TRAAK-Phosphatidylserine Interactions.

TRAAK and TREK2 are two-pore domain K+ (K2P) channels and are modulated by diverse factors including temperature, membrane stretching, and lipids, such as phosphatidic acid. In addition, copper and zinc, both of which are essential for life, are known to regulate TREK2 and a number of other ion channels. However, the role of ions in the association of lipids with integral membrane proteins is poorly understood. Here, we discover cupric ions selectively modulate the binding of phosphatidylserine (PS) to TRAAK but not TREK2. Other divalent cations (Ca2+, Mg2+, and Zn2+) bind both channels but have no impact on binding PS and other lipids. Additionally, TRAAK binds more avidly to Cu2+ and Zn2+ than TREK2. In the presence of Cu2+, TRAAK binds similarly to PS with different acyl chains, indicating a crucial role of the serine headgroup in coordinating Cu2+. High-resolution native mass spectrometry (MS) enables the determination of equilibrium binding constants for distinct Cu2+-bound stoichiometries and uncovered the highest coupling factor corresponds to a 1:1 PS-to-Cu2+ ratio. Interestingly, the next three highest coupling factors had a ∼1.5:1 PS-to-Cu2+ ratio. Our findings bring forth the role of cupric ions as an essential cofactor in selective TRAAK-PS interactions.

Probing heavy metal binding to phycobiliproteins

Blue‐green algae, also known as cyanobacteria, contain some of the most efficient light‐harvesting complexes known. These large, colourful complexes consist of phycobiliproteins which are extremely valuable in the cosmetics, food, nutraceutical and pharmaceutical industries. Additionally, the colourful and fluorescent properties of phycobiliproteins can be modulated by metal ions, making them highly attractive as heavy metal sensors and heavy metal scavengers. Although the overall quenching ability metal ions have on phycobiliproteins is known, the mechanism of heavy metal binding to phycobiliproteins is not fully understood, limiting their widespread quantitative applications. Here, we show using high‐resolution native mass spectrometry that phycobiliprotein complexes bind metal ions in different manners. Through monitoring the binding equilibria and metal‐binding stoichiometry, we show in particular copper and silver to have drastic, yet different effects on phycobiliprotein structure, both copper and silver modulate the overall complex properties. Together, the data reveals the mechanisms by which metal ions can modulate phycobiliprotein properties which can be used as a basis for the future design of metal‐related phycobiliprotein applications.

Multiplexed Screening of Thousands of Natural Products for Protein-Ligand Binding in Native Mass Spectrometry.

The structural diversity of natural products offers unique opportunities for drug discovery, but challenges associated with their isolation and screening can hinder the identification of drug-like molecules from complex natural product extracts. Here we introduce a mass spectrometry-based approach that integrates untargeted metabolomics with multistage, high-resolution native mass spectrometry to rapidly identify natural products that bind to therapeutically relevant protein targets. By directly screening crude natural product extracts containing thousands of drug-like small molecules using a single, rapid measurement, we could identify novel natural product ligands of human drug targets without fractionation. This method should significantly increase the efficiency of target-based natural product drug discovery workflows.

High-Resolution Native Mass Spectrometry.

Native mass spectrometry (MS) involves the analysis and characterization of macromolecules, predominantly intact proteins and protein complexes, whereby as much as possible the native structural features of the analytes are retained. As such, native MS enables the study of secondary, tertiary, and even quaternary structure of proteins and other biomolecules. Native MS represents a relatively recent addition to the analytical toolbox of mass spectrometry and has over the past decade experienced immense growth, especially in enhancing sensitivity and resolving power but also in ease of use. With the advent of dedicated mass analyzers, sample preparation and separation approaches, targeted fragmentation techniques, and software solutions, the number of practitioners and novel applications has risen in both academia and industry. This review focuses on recent developments, particularly in high-resolution native MS, describing applications in the structural analysis of protein assemblies, proteoform profiling of—among others—biopharmaceuticals and plasma proteins, and quantitative and qualitative analysis of protein–ligand interactions, with the latter covering lipid, drug, and carbohydrate molecules, to name a few.

Discrepancies between High-Resolution Native and Glycopeptide-Centric Mass Spectrometric Approaches: A Case Study into the Glycosylation of Erythropoietin Variants.

Glycosylation represents a critical quality attribute modulating a myriad of physiochemical properties and effector functions of biotherapeutics. Furthermore, a rising landscape of glycosylated biotherapeutics including biosimilars, biobetters, and fusion proteins harboring complicated and dynamic glycosylation profiles requires tailored analytical approaches capable of characterizing their heterogeneous nature. In this work, we perform in-depth evaluation of the glycosylation profiles of three glycoengineered variants of the widely used biotherapeutic erythropoietin. We analyzed these samples in parallel using a glycopeptide-centric liquid chromatography/mass spectrometry approach and high-resolution native mass spectrometry. Although for all of the studied variants the glycopeptide and native mass spectrometry data were in good qualitative agreement, we observed substantial quantitative differences arising from ionization deficiencies and unwanted neutral losses, in particular, for sialylated glycopeptides in the glycoproteomics approach. However, the latter provides direct information about glycosite localization. We conclude that the combined parallel use of native mass spectrometry and bottom-up glycoproteomics offers superior characterization of glycosylated biotherapeutics and thus provides a valuable attribute in the characterization of glycoengineered proteins and other complex biotherapeutics.

Adeno-associated virus capsid assembly is divergent and stochastic

Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-enveloped T = 1 icosahedral capsid of ~3.8 megaDalton, consisting of 60 subunits of 3 distinct viral proteins (VPs), which vary only in their N-terminus. While all three VPs play a role in cell-entry and transduction, their precise stoichiometry and structural organization in the capsid has remained elusive. Here we investigate the composition of several AAV serotypes by high-resolution native mass spectrometry. Our data reveal that the capsids assemble stochastically, leading to a highly heterogeneous population of capsids of variable composition, whereby even the single-most abundant VP stoichiometry represents only a small percentage of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition. The systematic scoring of the simulations against experimental native MS data offers a sensitive new method to characterize these therapeutically important heterogeneous capsids.

Glycoproteoform Profiles of Individual Patients' Plasma Alpha-1-Antichymotrypsin are Unique and Extensively Remodeled Following a Septic Episode.

Sepsis and septic shock remain the leading causes of death in intensive care units (ICUs), yet the pathogenesis originating from the inflammatory response during sepsis remains ambiguous. Acute-phase proteins are typically highly glycosylated, and the nature of the glycans have been linked to the incidence and severity of such inflammatory responses. To further build upon these findings we here monitored, the longitudinal changes in the plasma proteome and, in molecular detail, glycoproteoform profiles of alpha-1-antichymotrypsin (AACT) extracted from plasma of ten individual septic patients. For each patient we included four different time-points, including post-operative (before sepsis) and following discharge from the ICU. We isolated AACT from plasma depleted for albumin, IgG and serotransferrin and used high-resolution native mass spectrometry to qualitatively and quantitatively monitor the multifaceted glycan microheterogeneity of desialylated AACT, which allowed us to monitor how changes in the glycoproteoform profiles reflected the patient’s physiological state. Although we observed a general trend in the remodeling of the AACT glycoproteoform profiles, e.g. increased fucosylation and branching/LacNAc elongation, each patient exhibited unique features and responses, providing a resilient proof-of-concept for the importance of personalized longitudinal glycoproteoform profiling. Importantly, we observed that the AACT glycoproteoform changes induced by sepsis did not readily subside after discharge from ICU.

Molecular Mechanism of ISC Iron-Sulfur Cluster Biogenesis Revealed by High-Resolution Native Mass Spectrometry.

Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for many important biological processes including oxidative respiration, nitrogen fixation, and photosynthesis. Biosynthetic pathways assemble Fe-S clusters with different iron-to-sulfur stoichiometries and distribute these clusters to appropriate apoproteins. In the ISC pathway, the pyridoxal 5'-phosphate-dependent cysteine desulfurase enzyme IscS provides sulfur to the scaffold protein IscU, which templates the Fe-S cluster assembly. Despite their functional importance, mechanistic details for cluster synthesis have remained elusive. Recent advances in native mass spectrometry (MS) have allowed proteins to be preserved in native-like structures and support applications in the investigation of protein structure, dynamics, ligand interactions, and the identification of protein-associated intermediates. Here, we prepared samples under anaerobic conditions and then applied native MS to investigate the molecular mechanism for Fe-S cluster synthesis. This approach was validated by the high agreement between native MS and traditional visible circular dichroism spectroscopic assays. Time-dependent native MS experiments revealed potential iron- and sulfur-based intermediates that decay as the [2Fe-2S] cluster signal developed. Additional experiments establish that (i) Zn(II) binding stabilizes IscU and protects the cysteine residues from oxidation, weakens the interactions between IscU and IscS, and inhibits Fe-S cluster biosynthesis; and (ii) Fe(II) ions bind to the IscU active site cysteine residues and another lower affinity binding site and promote the intermolecular sulfur transfer reaction from IscS to IscU. Overall, these results support an iron-first model for Fe-S cluster synthesis and highlight the power of native MS in defining protein-associated intermediates and elucidating mechanistic details of enzymatic processes.

Drug Loading and Distribution of ADCs After Reduction or IdeS Digestion and Reduction.

High-resolution native mass spectrometry (MS) provides accurate mass measurements (within 30ppm) of intact ADCs and can also yield drug load distribution (DLD) and average drug to antibody ratio (DAR) in parallel with hydrophobic interaction chromatography (HIC). Native MS is furthermore unique in its ability to simultaneously detect covalent and noncovalent species in a mixture and for HIC peak identity assessment offline or online.As an orthogonal method described in this chapter, LC-MS following ADC reduction or IdeS (Fabricator) digestion and reduction can also be used to measure the DLD of light chain and Fd fragments for hinge native cysteine residues such as brentuximab vedotin. Both methods allow also the measurement of average DAR for both monomeric and multimeric species. In addition, the Fc fragments can be analyzed in the same run, providing a complete glycoprofile and the demonstration or absence of additional conjugation of this subdomain involved in FcRn and Fc-gammaR binding.

Intrinsic GTPase Activity of K-RAS Monitored by Native Mass Spectrometry

Mutations in RAS are associated with many different cancers and have been a therapeutic target for more than three decades. RAS cycles from an active to inactive state by both intrinsic and GTPase-activating protein (GAP)-stimulated hydrolysis. The activated enzyme interacts with downstream effectors, leading to tumor proliferation. Mutations in RAS associated with cancer are insensitive to GAP, and the rate of inactivation is limited to their intrinsic hydrolysis rate. Here, we use high-resolution native mass spectrometry (MS) to determine the kinetics and transition state thermodynamics of intrinsic hydrolysis for K-RAS and its oncogenic mutants. MS data reveal heterogeneity where both 2'-deoxy and 2'-hydroxy forms of GDP (guanosine diphosphate) and GTP (guanosine triphosphate) are bound to the recombinant enzyme. Intrinsic GTPase activity is directly monitored by the loss in mass of K-RAS bound to GTP, which corresponds to the release of phosphate. The rates determined from MS are in direct agreement with those measured using an established solution-based assay. Our results show that the transition state thermodynamics for the intrinsic GTPase activity of K-RAS is both enthalpically and entropically unfavorable. The oncogenic mutants G12C, Q61H, and G13D unexpectedly exhibit a 2'-deoxy GTP intrinsic hydrolysis rate higher than that for GTP.

Quantitative Determination of Protein-Ligand Affinity by Size Exclusion Chromatography Directly Coupled to High-Resolution Native Mass Spectrometry.

High throughput protein-ligand interaction screening assays employing mass spectrometric detection are widely used in early stage drug discovery. Mass spectrometry-based screening approaches employ a target protein added to a pool of small-molecule compounds, and binding is assessed by measuring ligands denatured from the complexes. Direct analysis of protein-ligand interactions using native mass spectrometry has been demonstrated but is not widely used due to the detection limit on protein size, the requirement of volatile buffers, and the necessity for specialized instrumentation to preserve weak interactions under native conditions. Here we present a robust, quantitative, and automated online size-exclusion chromatography-native mass spectrometry (SEC-nMS) platform for measuring affinities of noncovalent protein-small-molecule interactions on an Orbitrap mass spectrometer. Indoleamine 2,3-dioxygenase 1, a catabolic enzyme, and inhibitory ligands were employed as a demonstration of the method. Efficient separation and elution enabled preservation of protein-ligand complexes and increased throughput. The high sensitivity and intra charge state resolution at high m/ z offered by the Exactive Plus EMR Orbitrap allowed for protein ligand affinity quantitation and resolved individual compounds close in mass. Vc50 values determined via collision-induced dissociation experiments enabled the evaluation of complex stability in the gas phase and were found to be independent of the extent of complex formation. For the first time, Vc50 determinations were achieved on an inline SEC-nMS platform. Systematic comparison of our method with optimized chip-based nanoelectrospray infusion served as a reference for ligand screening and affinity quantitation and further revealed the advantages of SEC-MS.

Direct quality control of glycoengineered erythropoietin variants

Recombinant production of glycoprotein therapeutics like erythropoietin (EPO) in mammalian CHO cells rely on the heterogeneous N-glycosylation capacity of the cell. Recently, approaches for engineering the glycosylation capacities of mammalian cells for custom designed glycoforms have been developed. With these opportunities there is an increasing need for fast, sensitive, and global analysis of the glycoproteoform landscape produced to evaluate homogeneity and consistency. Here we use high-resolution native mass spectrometry to measure the glycoproteoform profile of 24 glycoengineered variants of EPO. Based on the unique mass and intensity profiles of each variant, we classify them according to similarities in glycosylation profiles. The classification distinguishes EPO variants with varying levels of glycan branchingand sialylation, which are crucial parameters in biotherapeutic efficacy. We propose that our methods could be of great benefit in the characterization of other glycosylated biopharmaceuticals, ranging from the initial clonal selection to batch-to-batch controls, and the assessment of similarity between biosimilar/biobetter products.