Resolving the micro-heterogeneity and structural integrity of monoclonal antibodies by hybrid mass spectrometric approaches

For therapeutic monoclonal antibodies (mAbs), detailed analysis of the structural integrity and heterogeneity, which results from multiple types of post-translational modifications (PTMs), is relevant to various processes, including product characterization, storage stability and quality control. Despite the recent rapid development of new bioanalytical techniques, it is still challenging to completely characterize the proteoform profile of a mAb. As a nearly indispensable tool in mAb analysis, mass spectrometry (MS) provides unique structural information at multiple levels. Here, we tested a hybrid strategy for the comprehensive characterization of micro-heterogeneity by integrating 2 state-of-the-art MS-based approaches, high-resolution native MS and targeted glycan profiling, to perform complementary analysis at the intact protein level and released glycan level, respectively. We compared the performance of these methods using samples of engineered half-body IgG4s and a panel of mAbs approved for human use. The glycosylation characterization data derived from these approaches were found to be mutually consistent in composition profiling, and complementary in identification and relative-quantitation of low-abundant uncommon glycoforms. In addition, multiple other sources of micro-heterogeneity, such as glycation, lack of glycosylation, and loss of light chains, could be detected by this approach, and the contribution of multiple types of modifications to the overall micro-heterogeneity could be assessed using our superposition algorithm. Our data demonstrate that the hybrid strategy allows reliable and thorough characterization of mAbs, revealing product characteristics that would easily be missed if only a single approach were used.     

Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.     

Three-Dimensional Structure and Biophysical Characterization of Staphylococcus aureus Cell Surface Antigen–Manganese Transporter MntC

MntC is a metal-binding protein component of the Mn^2 +-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensional structure of the protein was solved by X-ray crystallography at 2.2 Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn^2 +-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn^2 +-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium–hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn^2 +.     

The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst

Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. The scaffolding proteins are essential to procapsid assembly. Here, we describe the solution-based biophysical and functional characterization of the bacteriophage lambda (λ) scaffolding protein gpNu3. The purified protein possesses significant α-helical structure and appears to be partially disordered. Thermally induced denaturation studies indicate that secondary structures are lost in a cooperative, apparent two-state transition (T_m = 40.6 ± 0.3 °C) and that unfolding is, at least in part, reversible. Analysis of the purified protein by size-exclusion chromatography suggests that gpNu3 is highly asymmetric, which contributes to an abnormally large Stokes radius. The size-exclusion chromatography data further indicate that the protein self-associates in a concentration-dependent manner. This was confirmed by analytical ultracentrifugation studies, which reveal a monomer-dimer equilibrium (K_d,app ~ 50 μM) and an asymmetric protein structure at biologically relevant concentrations. Purified gpNu3 promotes the polymerization of gpE, the λ major capsid protein, into virus-like particles that possess a native-like procapsid morphology. The relevance of this work with respect to procapsid assembly in the complex double-stranded DNA viruses is discussed.     

Organization of interphase chromatin

The organization of interphase chromatin spans many topics, ranging in scale from the molecular level to the whole nucleus, and its study requires a concomitant range of experimental approaches. In this review, we examine these approaches, the results they have generated, and the interfaces between them. The greatest challenge appears to be the integration of information on whole nuclei obtained by light microscopy with data on nucleosome–nucleosome interactions and chromatin higher-order structures, obtained in vitro using biophysical characterization, atomic force microscopy, and electron microscopy. We consider strategies that may assist in the integration process, and we review emerging technologies that promise to reduce the “resolution gap.” This article is dedicated to the memory of Hans Ris.     

Immunization method for multi-pass membrane proteins using highly metastatic cell lines

A novel method using metastatic breast cancer cell lines was established for producing monoclonal antibodies (mAbs) against multi-span membrane proteins. Grafting of metastatic cells (MCF7-14) into the mammary gland of BALB/cJ/nu/nu mice induced splenic hypertrophy (1.6–3.0 × 10⁸ cells/spleen [n = 6]). More than half of the mAbs against MCF7-14 cells reacted with the cell membrane. Inducing production of antibodies against the extracellular domain of multi-pass membrane proteins is difficult. Because the protein structure becomes more complex as the number of transmembrane domains increases, preparing antigens for immunization in which the original structure is maintained is challenging. Using highly metastatic MDA-MB231 cells as the host cell line, we produced mAbs against a 12 transmembrane protein, solute carrier family 6 member 6 (SLC6A6), as a model antigen. When SLC6A6-overexpressing MDA-MB231 cells were grafted into nude mice, the number of splenocytes increased to 2.7–11.4 × 10⁸ cells/spleen (n = 10). Seven mAb-producing clones that not only recognized the extracellular domain of SLC6A6 but also were of the IgG subclass were obtained. Immunocytochemistry and flow cytometry analyses revealed that these mAbs recognized the native form of the extracellular domain of SLC6A6 on the cell surface. Our novel immunization method involving highly metastatic cells could be used to develop therapeutic mAbs against other multi-pass membrane proteins.     

Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1

We have characterized the structures of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E₂ synthase-1 (mPGES-1) in intact cells using bifunctional and photo-activatable crosslinking agents. A dimeric complex was detected for COX-2 by both crosslinking approaches, consistent with the crystal structure of the enzyme. For mPGES-1, treatment of A549 cells with disuccinimidyl suberate yielded immunoreactive protein bands corresponding to a dimer (33 kDa) and a trimer (45 kDa), as observed for the isolated enzyme. Photo-crosslinking with photoactivatable methionine in intact cells generated complexes with molecular weights corresponding to the dimer (33 kDa) and two putative trimer forms (50 and 55 kDa). Treatment with the selective mPGES-1 inhibitor MF63 prevented the formation of the 50 and 55 kDa crosslinked complexes, while an inactive structural analogue had no effect. Our data indicate that COX-2 forms a dimer in intact cells and that mPGES-1 has an oligomeric structure that can be disrupted by a selective inhibitor.     

Zinc induced folding is essential for TIM15 activity as an mtHsp70 chaperone

Background

TIM15/Zim17 in yeast and its mammalian ortholog Hep are Zn^2 + finger (Cys4) proteins that assist mtHsp70 in protein import into the mitochondrial matrix.

Methods

Here we characterized the Zn^2 + induced TIM15 folding integrating biophysical and computational approaches.

Results

TIM15 folding occurs from an essentially unstructured conformation to a Zn^2 +-coordinated protein in a fast and markedly temperature-dependent process. Moreover, we demonstrate unambiguously that Zn^2 + induced TIM15 folding is essential for its role as mtHsp70 chaperone since in the unstructured apo state TIM15 does not bind to mtHsp70 and is unable to prevent its aggregation. Molecular dynamics simulations help to understand the crucial role of Zn^2 + in promoting a stable and functional 3D architecture in TIM15. It is shown that the metal ion, through its coordinating cysteine residues, can mediate relevant long-range effects with the interaction interface for mtHsp70 coupling thus folding and function.

Conclusions

Zn^2 + induced TIM15 folding is essential for its function and likely occurs in mitochondrial matrix where high concentrations of Zn^2 + were reported.

General significance

The combination of experimental and computational approaches presented here provide an integrated structural, kinetic and thermodynamic view of the folding of a mitochondrial zinc finger protein, which might be relevant to understand the organelle import of proteins sharing this fold.     

Errata

Abstract

Mass spectrometry (MS)-based proteomics is an unrivaled tool for studying complex biological systems and diseases in the post-genomic era. In recent years, MS has emerged as a powerful structural biological tool to characterize protein conformation and conformational dynamics. The advantages of MS in structural studies are most evident for membrane proteins such as GPCRs (G protein-coupled receptors), where other well-established structural methods such as X-ray crystallography and NMR remain challenging. For proteins with available high-resolution structures, MS-based structural strategies can provide valuable, previously inaccessible information on protein conformational changes and dynamics, protein motion/flexibility, ligand–protein binding, and protein–protein interfaces. In the past several years, we have developed and adapted a number of MS-based structural approaches, such as CDSiL-MS (Conformational changes and Dynamics using Stable-isotope Labeling and MS), CXMS (Crosslinking/MS) and HDXMS (Hydrogen-Deuterium Exchange MS), to study protein structures and conformational dynamics in human β2-adrenegic receptor (β2AR) signaling. In this mini-review, we will highlight several examples demonstrating the power of MS in structural analysis to better elucidate the structural basis of GPCR signaling, particularly through the β-arrestin-mediated GPCR signaling pathway.     

Effective reversed-phase ion pair high-performance liquid chromatography method for the separation and characterization of intact low-molecular-weight heparins

A simple, selective, and efficient reversed-phase ion pair high-performance liquid chromatography (RPIP-HPLC) method was developed for the separation of various commercially available intact low-molecular-weight heparins (LMWHs). The developed method uses a C₁₈ column (150 × 4.6 mm) with diode array detection at 230 nm, flow rate at 1.0 ml/min, and a mobile phase containing acetonitrile/water (32:68%), tetrabutylammonium hydroxide (15 mM), and ammonium acetate (50 mM) at pH 7.0. The performance of this method was assessed in terms of selectivity, linearity, intra- and interday precision, and accuracy. The novel application of RPIP-HPLC with evaporative light scattering detection (ELSD) for the analysis of intact LMWHs was demonstrated. Intact LMWHs were analyzed with superior resolution and peak shape. Different chromatographic profiles were obtained for different LMWHs showing significant structural diversity. This method clearly showed chemical changes that occurred to LMWH under the stress condition. This method can be applied for the separation, identification, characterization, and pharmaceutical stability analysis of various LMWHs.     

An expression and purification system for the biosynthesis of adenosine receptor peptides for biophysical and structural characterization

Biophysical and structural characterization of G protein-coupled receptors (GPCRs) has been limited due to difficulties in expression, purification, and vitro stability of the full-length receptors. "Divide and conquer" approaches aimed at the NMR characterization of peptides corresponding to specific regions of the receptor have yielded insights into the structure and dynamics of GPCR activation and signaling. Though significant progress has been made in the generation of peptides that are composed of GPCR transmembrane domains, current methods utilize fusion protein strategies that require chemical cleavage and peptide separation via chromatographic means. We have developed an expression and purification system based on fusion to ketosteroid isomerase, thrombin cleavage, and tandem affinity chromatography that enables the solubilization, cleavage, and characterization in a single detergent system relevant for biophysical and structural characterization. We have applied this expression and purification system to the production and characterization of peptides of the adenosine receptor family of GPCRs in Escherichia coli. Herein, we demonstrate using a model peptide that includes extracellular loop 3, transmembrane domain 7, and a portion of the carboxy-terminus of the adenosine A(2)a receptor that the peptide is sufficiently pure for biophysical characterization, where it adopts α-helical structure. Furthermore, we demonstrate the utility of this system by optimizing the construct for thrombin processing and apply the system to peptides with more complex structures.     

Comparison and functional implications of the 3D architectures of viral tRNA-like structures

RNA viruses co-opt the host cell's biological machinery, and their infection strategies often depend on specific structures in the viral genomic RNA. Examples are tRNA-like structures (TLSs), found at the 3′ end of certain plant viral RNAs, which can use the cell's aminoacyl tRNA-synthetases (AARSs) to drive addition of an amino acid to the 3′ end of the viral RNA. TLSs are multifunctional RNAs involved in processes such as viral replication, translation, and viral RNA stability; these functions depend on their fold. Experimental result-based structural models of TLSs have been published. In this study, we further examine these structures using a combination of biophysical and biochemical approaches to explore the three-dimensional (3D) architectures of TLSs from the turnip yellow mosaic virus (TYMV), tobacco mosaic virus (TMV), and brome mosaic virus (BMV). We find that despite similar function, these RNAs are biophysically diverse: the TYMV TLS adopts a characteristic tRNA-like L shape, the BMV TLS has a large compact globular domain with several helical extensions, and the TMV TLS aggregates in solution. Both the TYMV and BMV TLS RNAs adopt structures with tight backbone packing and also with dynamic structural elements, suggesting complexities and subtleties that cannot be explained by simple tRNA mimicry. These results confirm some aspects of existing models and also indicate how these models can be improved. The biophysical characteristics of these TLSs show how these multifunctional RNAs might regulate various viral processes, including negative strand synthesis, and also allow comparison with other structured RNAs.     

Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli

The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with ¹³C, ¹⁵N, ²H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.     

Antigen-antibody interface properties: Composition, residue interactions, and features of 53 non-redundant structures

The structures of protein antigen-antibody (Ag-Ab) interfaces contain information about how Ab recognize Ag as well as how Ag are folded to present surfaces for Ag recognition. As such, the Ab surface holds information about Ag folding that resides with the Ab-Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, a data set comprised of 53 non-redundant 3D structures of Ag-Ab complexes was analyzed. We assessed the physical and biochemical features of the Ag-Ab interfaces and the degree to which favored interactions exist between amino acid residues on the corresponding interface surfaces. Amino acid compositional analysis of the interfaces confirmed the dominance of TYR in the Ab paratope-containing surface (PCS), with almost two fold greater abundance than any other residue. Additionally TYR had a much higher than expected presence in the PCS compared to the surface of the whole antibody (defined as the occurrence propensity), along with aromatics PHE, TRP, and to a lesser degree HIS and ILE. In the Ag epitope-containing surface (ECS), there were slightly increased occurrence propensities of TRP and TYR relative to the whole Ag surface, implying an increased significance over the compositionally most abundant LYS > ASN > GLU > ASP > ARG. This examination encompasses a large, diverse set of unique Ag-Ab crystal structures that help explain the biological range and specificity of Ag-Ab interactions. This analysis may also provide a measure of the significance of individual amino acid residues in phage display analysis of Ag binding.     

Channelrhodopsin unchained: Structure and mechanism of a light-gated cation channel

The new and vibrant field of optogenetics was founded by the seminal discovery of channelrhodopsin, the first light-gated cation channel. Despite the numerous applications that have revolutionised neurophysiology, the functional mechanism is far from understood on the molecular level. An arsenal of biophysical techniques has been established in the last decades of research on microbial rhodopsins. However, application of these techniques is hampered by the duration and the complexity of the photoreaction of channelrhodopsin compared with other microbial rhodopsins. A particular interest in resolving the molecular mechanism lies in the structural changes that lead to channel opening and closure. Here, we review the current structural and mechanistic knowledge that has been accomplished by integrating the static structure provided by X-ray crystallography and electron microscopy with time-resolved spectroscopic and electrophysiological techniques. The dynamical reactions of the chromophore are effectively coupled to structural changes of the protein, as shown by ultrafast spectroscopy. The hierarchical sequence of structural changes in the protein backbone that spans the time range from 10^− 12 s to 10^− 3 s prepares the channel to open and, consequently, cations can pass. Proton transfer reactions that are associated with channel gating have been resolved. In particular, glutamate 253 and aspartic acid 156 were identified as proton acceptor and donor to the retinal Schiff base. The reprotonation of the latter is the critical determinant for channel closure. The proton pathway that eventually leads to proton pumping is also discussed. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

A gas chromatographic method for quantification of detergents frequently used in membrane protein structural studies

A novel and reliable gas chromatography-flame ionization detection (GC-FID) method that can separate and quantify detergents frequently used in membrane protein structural studies has been developed. Different detergents were identified through FID peaks with different retention times. A quadratic regression curve was found to fit the integrated FID peak area against different detergent concentrations. Detergents can be quantified as low as the nanogram level: lauryl-dimethylamine-N-oxide (LDAO), 5 ng; dodecyl maltoside (DDM), 10 ng; and dodecyl phosphocholine (DPC), 50 ng. This method can be applied directly to measure detergent concentration and molar ratio of membrane protein to detergents during membrane protein extraction, purification, concentration, and crystallization.     

Biophysical cues enhance myogenesis of human adipose derived stem/stromal cells

Adipose-derived stem/stromal cell (ASC)-based tissue engineered muscle grafts could provide an effective alternative therapy to autografts – which are limited by their availability – for the regeneration of damaged muscle. However, the current myogenic potential of ASCs is limited by their low differentiation efficiency into myoblasts. The aim of this study was to enhance the myogenic response of human ASCs to biochemical cues by providing biophysical stimuli (11% cyclic uniaxial strain, 0.5 Hz, 1 h/day) to mimic the cues present in the native muscle microenvironment. ASCs elongated and fused upon induction with myogenic induction medium alone. Yet, their myogenic characteristics were significantly enhanced with the addition of biophysical stimulation; the nuclei per cell increased approximately 4.5-fold by day 21 in dynamic compared to static conditions (23.3 ± 7.3 vs. 5.2 ± 1.6, respectively), they aligned at almost 45° to the direction of strain, and exhibited significantly higher expression of myogenic proteins (desmin, myoD and myosin heavy chain). These results demonstrate that mimicking the biophysical cues inherent to the native muscle microenvironment in monolayer ASC cultures significantly improves their differentiation along the myogenic lineage.