Novel Analytical Methods Combining Non-Denaturing Chromatography and Mass Spectrometry to Study Biopolymer Structure and Interactions

Biotherapeutics, an emerging class of medicines containing biopolymers (e.g., proteins, peptide, and polysaccharides) have been developed for a variety of indications including cancer, autoimmune, genetic, and blood diseases. Among these biopolymers protein therapeutics have been the rapidly growing segment in the pharmaceutical industry. This trend combined with the complexity of proteins has necessitated the development of powerful and robust analytical methods to study their structure and interactions with physiological partners. Mass spectrometry (MS) has become an indispensable tool to analyze various attributes of protein drugs such as profiling of intact mass, amino acid sequencing, and post translational modifications (PTMs). In this work novel analytical methods have been developed by combining non-denaturing chromatographic separations (such as size exclusion chromatography and ion exchange chromatography) with native ESI MS. In addition to providing information of mass and non-covalent assemblies, native ESI MS has an advantage of probing conformational integrity of proteins by analyzing ionic charge state distributions in the mass spectrum. Size exclusion chromatography with online native ESI MS detection method developed in this work allowed characterization of higher order structure of proteins and probe aggregation propensity. Using the same SEC/native ESI MS workflow a method to find kinetics and equilibrium binding constant of transient protein interactions have also been developed. Combination of ion exchange chromatography (IXC) with native ESI MS and MS/MS detection was developed and shown to be effective in characterization of positional isomers of protein conjugates and PTMs of a biotherapeutic recombinant human interferon beta 1a. To characterize highly heterogeneous PEGylated glycoprotein (conjugated with a 20kDa PEG) analytical methods using IXC/native ESI MS combined with limited charge reduction and collisional activation were developed

High Pressure Liquid Chromatography Studies of the Reaction of Platinum Complexes with Peptides

Platinum complexes (cisplatin, carboplatin and oxaliplatin) are effective anticancer agents. However the major drawbacks of platinum chemotherapy are toxic side effects and resistance. The affinity of platinum complexes to sulfur donor ligands of side chains of methionine and cysteine amino acids was assumed to be responsible for toxicity and resistance. Recently, it was found that the reaction of platinum complex with proteins containing sulfur donor ligands could actually favor its anticancer activity. Copper transporter 1 (Ctr 1), a protein involved in the transport of copper into the cell, also helps in the influx of cisplatin by binding to N-terminal domain of Ctr 1 which is rich in methionine and histidine residues. A better understanding of how the size and shape of amine ligand, and leaving groups affect the reaction of platinum (II) complexes with methionine could give new ways to optimize its anticancer activity. This preliminary research focuses to answer this by HPLC-UV-VIS analysis of bulky platinum complexes including [Pt(dien)Cl]Cl, Pt(Me4en)(NO3)2 and Pt(en)(NO3)2 with two methionine containing small peptides that serve as models for protein interactions

Matrix Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry: An Imaging Modality to Monitor the Effects of Gene Therapy in a Murine Model of GM1 Gangliosidosis

GM1 gangliosidosis is an autosomal recessive lysosomal storage disorder caused by an enzyme deficiency of β-galactosidase (β-gal) leading to toxic accumulation of GM1 ganglioside in the central nervous system (CNS) and progressive neurodegeneration. Adeno-associated virus (AAV) mediated gene delivery of lysosomal enzymes to the CNS has shown great potential for the treatment of lysosomal storage diseases with neurological involvement. In this work we use MALDI mass spectrometry imaging (MSI) to assess the spatial distribution of gangliosides, ganglioside metabolites and related lipids in a GM1 gangliosidosis mouse brain model following adeno-associated virus (AAV) gene therapy. Deficiency of β-galactosidase enzyme in a β-gal-/- mouse brain showed an overall 8-fold increase in GM1 relative to the control by MSI analysis, with specific spatial localization based on its ceramide content. Bilateral thalamic injection of AAVrh10-bgal in β-gal-/- mice significantly reduced GM1 levels relative to untreated β-gal-/- mice. The therapeutic efficacy of this approach is through distribution of functional enzyme via axonal transport through the extensive connectivity of the thalamus with most of the brain, with some exceptions such as the temporal cortex. Accordingly MSI showed AAV gene therapy reduced GM1 nearly to the control levels in all regions of the brain except in the temporal cerebral cortex. This correlated with low levels of bgal in this brain region as assessed by histochemical staining of tissue sections. MSI also detected asialo-GM1 and other ganglioside metabolites elevated in untreated β-gal-/- mice, which were also reduced after AAV therapy. Interestingly sulfated galactocereberosides reduced in the myelin sheath in untreated β-gal-/- mice were restored to normal levels after AAV therapy. Overall, this study demonstrates that MALDI MSI can be used to map specific target analytes and their metabolites while also offering the ability to detect unanticipated effects caused by gene therapy

High Pressure Liquid Chromatography Studies of the Reaction of Platinum Complexes with Peptides

Platinum complexes (cisplatin, carboplatin and oxaliplatin) are effective anticancer agents. However the major drawbacks of platinum chemotherapy are toxic side effects and resistance. The affinity of platinum complexes to sulfur donor ligands of side chains of methionine and cysteine amino acids was assumed to be responsible for toxicity and resistance. Recently, it was found that the reaction of platinum complex with proteins containing sulfur donor ligands could actually favor its anticancer activity. Copper transporter 1 (Ctr 1), a protein involved in the transport of copper into the cell, also helps in the influx of cisplatin by binding to N-terminal domain of Ctr 1 which is rich in methionine and histidine residues. A better understanding of how the size and shape of amine ligand, and leaving groups affect the reaction of platinum (II) complexes with methionine could give new ways to optimize its anticancer activity. This preliminary research focuses to answer this by HPLC-UV-VIS analysis of bulky platinum complexes including [Pt(dien)Cl]Cl, Pt(Me4en)(NO3)2 and Pt(en)(NO3)2 with two methionine containing small peptides that serve as models for protein interactions

Characterization of Intact Protein Conjugates and Biopharmaceuticals Using Ion-Exchange Chromatography with Online Detection by Native Electrospray Ionization Mass Spectrometry and Top-Down Tandem Mass Spectrometry

Characterization of biopharmaceutical products is a challenging task, which needs to be carried out at several different levels (including both primary structure and conformation). An additional difficulty frequently arises due to the structural heterogeneity inherent to many protein-based therapeutics (e.g., extensive glycosylation or “designer” modifications such as chemical conjugation) or introduced postproduction as a result of stress (e.g., oxidation and deamidation). A combination of ion-exchange chromatography (IXC) with online detection by native electrospray ionization mass spectrometry (ESI MS) allows characterization of complex and heterogeneous therapeutic proteins and protein conjugates to be accomplished at a variety of levels without compromising their conformational integrity. The IXC/ESI MS measurements allow protein conjugates to be profiled by analyzing conjugation stoichiometry and the presence of multiple positional isomers, as well as to establish the effect of chemical modifications on the conformational integrity of each species. While mass profiling alone is not sufficient for identification of nonenzymatic post-translational modifications (PTMs) that result in a very small mass change of the eluting species (e.g., deamidation), this task can be completed using online top-down structural analysis, as demonstrated using stressed interferon-β as an example. The wealth of information that can be provided by IXC/native ESI MS and tandem mass spectrometry (MS/MS) on protein-based therapeutics will undoubtedly make it a very valuable addition to the experimental toolbox of biopharmaceutical analysis

A conserved folding nucleus sculpts the free energy landscape of bacterial and archaeal orthologs from a divergent TIM barrel family

The amino acid sequences of proteins have evolved over billions of years, preserving their structures and functions while responding to evolutionary forces. Are there conserved sequence and structural elements that preserve the protein folding mechanisms? The functionally diverse and ancient (betaalpha)1-8 TIM barrel motif may answer this question. We mapped the complex six-state folding free energy surface of a approximately 3.6 billion y old, bacterial indole-3-glycerol phosphate synthase (IGPS) TIM barrel enzyme by equilibrium and kinetic hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS on the intact protein reported exchange in the native basin and the presence of two thermodynamically distinct on- and off-pathway intermediates in slow but dynamic equilibrium with each other. Proteolysis revealed protection in a small (alpha1beta2) and a large cluster (beta5alpha5beta6alpha6beta7) and that these clusters form cores of stability in Ia and Ibp The strongest protection in both states resides in beta4alpha4 with the highest density of branched aliphatic side chain contacts in the folded structure. Similar correlations were observed previously for an evolutionarily distinct archaeal IGPS, emphasizing a key role for hydrophobicity in stabilizing common high-energy folding intermediates. A bioinformatics analysis of IGPS sequences from the three superkingdoms revealed an exceedingly high hydrophobicity and surprising alpha-helix propensity for beta4, preceded by a highly conserved betaalpha-hairpin clamp that links beta3 and beta4. The conservation of the folding mechanisms for archaeal and bacterial IGPS proteins reflects the conservation of key elements of sequence and structure that first appeared in the last universal common ancestor of these ancient proteins

Characterization of Small Protein Aggregates and Oligomers Using Size Exclusion Chromatography with Online Detection by Native Electrospray Ionization Mass Spectrometry

Self-association of proteins is important in a variety of processes ranging from acquisition of native quaternary structure (where the association is tightly controlled and proceeds in a highly ordered fashion) to aggregation and amyloidosis. The latter is frequently accompanied (or indeed triggered) by the loss of the native structure, but a clear understanding of the complex relationship between conformational changes and protein self-association/aggregation remains elusive due to the great difficulty in characterizing these complex and frequently heterogeneous species. In this study, size exclusion chromatography (SEC) was used in combination with online detection by native electrospray ionization mass spectrometry (ESI MS) to characterize a commercial protein sample (serum albumin) that forms small aggregates. Although noncovalent dimers and trimers of this protein are readily detected by native ESI MS alone, combination of SEC and ESI MS allows a distinction to be made between the oligomers present in solution and those formed during the ESI process (artifacts of ESI MS). Additionally, native ESI MS detection allows a partial loss of conformation integrity to be detected across all albumin species present in solution. Finally, ESI MS detection allows these analyses to be carried out readily even in the presence of other abundant proteins coeluting with albumin. Native ESI MS as an online detection method for SEC also enables meaningful characterization of species representing different quaternary organization of a recombinant glycoprotein human arylsulfatase A even when their rapid interconversion prevents their separation on the SEC time scale

LC/MS at the whole protein level: Studies of biomolecular structure and interactions using native LC/MS and cross-path reactive chromatography (XP-RC) MS

Interfacing liquid chromatography (LC) with electrospray ionization (ESI) to enable on-line MS detection hadbeen initially implemented using reversed phase LC, which in the past three decades remained the default type ofchromatography used for LC/MS and LC/MS/MS studies of protein structure. In contrast, the advantages of othertypes of LC as front-ends for ESI MS, particularly those that allow biopolymer higher order structure to bepreserved throughout the separation process, enjoyed relatively little appreciation until recently. However, thepast few years witnessed a dramatic surge of interest in the so-called “native” (with “non-denaturing” beingperhaps a more appropriate adjective) LC/MS and LC/MS/MS analyses within the bioanalytical and biophysicalcommunities. This review focuses on recent advances in this field, with an emphasis on size exclusion and ionexchange chromatography as front-end platforms for protein characterization by LC/MS. Also discussed are thebenefits provided by the integration of chemical reactions in the native LC/MS analyses, including both ionchemistry in the gas phase (e.g., limited charge reduction for characterization of highly heterogeneous biopolymers)and solution-phase reactions (using the recently introduced technique cross-path reactive chromatography)

Immunopeptidome profiling of human coronavirus OC43-infected cells identifies CD4 T-cell epitopes specific to seasonal coronaviruses or cross-reactive with SARS-CoV-2.

Seasonal "common-cold" human coronaviruses are widely spread throughout the world and are mainly associated with mild upper respiratory tract infections. The emergence of highly pathogenic coronaviruses MERS-CoV, SARS-CoV, and most recently SARS-CoV-2 has prompted increased attention to coronavirus biology and immunopathology, but the T-cell response to seasonal coronaviruses remains largely uncharacterized. Here we report the repertoire of viral peptides that are naturally processed and presented upon infection of a model cell line with seasonal coronavirus OC43. We identified MHC-bound peptides derived from each of the viral structural proteins (spike, nucleoprotein, hemagglutinin-esterase, membrane, and envelope) as well as non-structural proteins nsp3, nsp5, nsp6, and nsp12. Eighty MHC-II bound peptides corresponding to 14 distinct OC43-derived epitopes were identified, including many at very high abundance within the overall MHC-II peptidome. Fewer and less abundant MHC-I bound OC43-derived peptides were observed, possibly due to MHC-I downregulation induced by OC43 infection. The MHC-II peptides elicited low-abundance recall T-cell responses in most donors tested. In vitro assays confirmed that the peptides were recognized by CD4+ T cells and identified the presenting HLA alleles. T-cell responses cross-reactive between OC43, SARS-CoV-2, and the other seasonal coronaviruses were confirmed in samples of peripheral blood and peptide-expanded T-cell lines. Among the validated epitopes, spike protein S903-917 presented by DPA1*01:03/DPB1*04:01 and S1085-1099 presented by DRB1*15:01 shared substantial homology to other human coronaviruses, including SARS-CoV-2, and were targeted by cross-reactive CD4 T cells. Nucleoprotein N54-68 and hemagglutinin-esterase HE128-142 presented by DRB1*15:01 and HE259-273 presented by DPA1*01:03/DPB1*04:01 are immunodominant epitopes with low coronavirus homology that are not cross-reactive with SARS-CoV-2. Overall, the set of naturally processed and presented OC43 epitopes comprise both OC43-specific and human coronavirus cross-reactive epitopes, which can be used to follow CD4 T-cell cross-reactivity after infection or vaccination, and to guide selection of epitopes for inclusion in pan-coronavirus vaccines

Succination inactivates gasdermin D and blocks pyroptosis

Activated macrophages undergo a metabolic switch to aerobic glycolysis accumulating Krebs cycle intermediates that alter transcription of immune response genes. Here we extend these observations by defining fumarate as an inhibitor of pyroptotic cell death. We found that dimethyl fumarate (DMF) delivered to cells or endogenous fumarate reacts with gasdermin D (GSDMD) at critical cysteine residues to form S-(2-succinyl)-cysteine. GSDMD succination prevents its interaction with caspases, limiting its processing, oligomerization, and capacity to induce cell death. In mice, the administration of DMF protects against LPS shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis (EAE) by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics including DMF used to treat multiple sclerosis