Mechanistic insights into complement pathway inhibition by CR1 domain duplication.

Complement receptor 1 (CR1) is a membrane glycoprotein with a highly duplicated domain structure able to bind multiple ligands such as C3b and C4b, the activated fragments of complement components C3 and C4, respectively. We have previously used our knowledge of this domain structure to identify CSL040, a soluble extracellular fragment of CR1 containing the long homologous repeat (LHR) domains A, B, and C. CSL040 retains the ability to bind both C3b and C4b but is also a more potent complement inhibitor than other recombinant CR1-based therapeutics. To generate soluble CR1 variants with increased inhibitory potential across all three complement pathways, or variants with activity skewed to specific pathways, we exploited the domain structure of CR1 further by generating LHR domain duplications. We identified LHR-ABCC, a soluble CR1 variant containing a duplicated C3b binding C-terminal LHR-C domain that exhibited significantly enhanced alternative pathway inhibitory activity in vitro compared to CSL040. Another variant, LHR-BBCC, containing duplications of both LHR-B and LHR-C with four C3b binding sites, was shown to have reduced classical/lectin pathway inhibitory activity compared to CSL040, but comparable alternative pathway activity. Interestingly, multiplication of the C4b-binding LHR-A domain resulted in only minor increases in classical/lectin pathway inhibitory activity. The CR1 duplication variants characterized in these in vitro potency assays, as well as in affinity in solution C3b and C4b binding assays, not only provides an opportunity to identify new therapeutic molecules, but also additional mechanistic insights to the multiple interactions between CR1 and C3b/C4b.

HDX-MS study on garadacimab binding to activated FXII reveals potential binding interfaces through differential solvent exposure.

Hageman factor (FXII) is an essential component in the intrinsic coagulation cascade and a therapeutic target for the prophylactic treatment of hereditary angioedema (HAE). CSL312 (garadacimab) is a novel high-affinity human antibody capable of blocking activated FXII activity that is currently undergoing Phase 3 clinical trials in HAE. Structural studies using hydrogen/deuterium exchange coupled to mass spectrometry revealed evidence of interaction between the antibody and regions surrounding the S1 specificity pocket of FXII, including the 99-loop, 140-loop, 180-loop, and neighboring regions. We propose complementarity-determining regions (CDRs) in heavy-chain CDR2 and CDR3 as potential paratopes on garadacimab, and the 99-loop, 140-loop, 180-loop, and 220-loop as binding sites on the beta chain of activated FXII (ß-FXIIa).

Plasma-Derived Hemopexin as a Candidate Therapeutic Agent for Acute Vaso-Occlusion in Sickle Cell Disease: Preclinical Evidence.

People living with sickle cell disease (SCD) face intermittent acute pain episodes due to vaso-occlusion primarily treated palliatively with opioids. Hemolysis of sickle erythrocytes promotes release of heme, which activates inflammatory cell adhesion proteins on endothelial cells and circulating cells, promoting vaso-occlusion. In this study, plasma-derived hemopexin inhibited heme-mediated cellular externalization of P-selectin and von Willebrand factor, and expression of IL-8, VCAM-1, and heme oxygenase-1 in cultured endothelial cells in a dose-responsive manner. In the Townes SCD mouse model, intravenous injection of free hemoglobin induced vascular stasis (vaso-occlusion) in nearly 40% of subcutaneous blood vessels visualized in a dorsal skin-fold chamber. Hemopexin administered intravenously prevented or relieved stasis in a dose-dependent manner. Hemopexin showed parallel activity in relieving vascular stasis induced by hypoxia-reoxygenation. Repeated IV administration of hemopexin was well tolerated in rats and non-human primates with no adverse findings that could be attributed to human hemopexin. Hemopexin had a half-life in wild-type mice, rats, and non-human primates of 80-102 h, whereas a reduced half-life of hemopexin in Townes SCD mice was observed due to ongoing hemolysis. These data have led to a Phase 1 clinical trial of hemopexin in adults with SCD, which is currently ongoing.

Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδ T cells.

Gamma delta (γδ) T cells are essential to protective immunity. In humans, most γδ T cells express Vγ9Vδ2+ T cell receptors (TCRs) that respond to phosphoantigens (pAgs) produced by cellular pathogens and overexpressed by cancers. However, the molecular targets recognized by these γδTCRs are unknown. Here, we identify butyrophilin 2A1 (BTN2A1) as a key ligand that binds to the Vγ9+ TCR γ chain. BTN2A1 associates with another butyrophilin, BTN3A1, and these act together to initiate responses to pAg. Furthermore, binding of a second ligand, possibly BTN3A1, to a separate TCR domain incorporating Vδ2 is also required. This distinctive mode of Ag-dependent T cell activation advances our understanding of diseases involving pAg recognition and creates opportunities for the development of γδ T cell-based immunotherapies.

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

Raw N-glycan mass spectrometry imaging data on formalin-fixed mouse kidney.

Provided is the annotated raw data for N-glycan mass spectrometry imaging (MSI) annotations in thin cross-sections of formalin-fixed and paraffin-embedded murine kidney. Relevant meta-data have been provided in this brief and the raw MSI data can be accessed using ProteomeXchange with the PRoteomics IDEntifications (PRIDE) identifier PXD009808. This brief is the first in a set of submissions from our group which will make raw data publicly accessible for existing and future MSI studies.

Phenotype-specific recombinant haptoglobin polymers co-expressed with C1r-like protein as optimized hemoglobin-binding therapeutics.

BACKGROUND: Preclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1-1 versus Hp 2-2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA. ProHp is proteolytically cleaved by complement C1r subcomponent-like protein (C1r-LP) in the endoplasmic reticulum. Two main allelic Hp variants, HP1 and HP2 exist. The larger HP2 is considered to be the ancestor variant of all human Hp alleles and is characterized by an α2-chain, which contains an extra cysteine residue that pairs with additional α-chains generating multimers with molecular weights of 200-900 kDa. The two human HP1 alleles (HP1F and HP1S) differ by a two-amino-acid substitution polymorphism within the α-chain and are derived from HP2 by recurring exon deletions. RESULTS: In the present study, we describe a process for the production of recombinant phenotype specific Hp polymers in mammalian FS293F cells. This approach demonstrates that efficient expression of mature and fully functional protein products requires co-expression of active C1r-LP. The functional characterization of our proteins, which included monomer/polymer distribution, binding affinities as well as NO-sparing and antioxidant functions, demonstrated that C1r-LP-processed recombinant Hp demonstrates equal protective functions as plasma derived Hp in vitro as well as in animal studies. CONCLUSIONS: We present a recombinant production process for fully functional phenotype-specific Hp therapeutics. The proposed process could accelerate the development of Hb scavengers to treat patients with cell-free Hb associated disease states, such as sickle cell disease and other hemolytic conditions.

MALDI imaging mass spectrometry of N-linked glycans on formalin-fixed paraffin-embedded murine kidney.

Recent developments in spatial proteomics have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed paraffin-embedded clinical tissue samples. This type of analysis is commonly referred to as matrix-assisted laser desorption/ionization (MALDI) imaging. Recently, formalin-fixed paraffin-embedded MALDI imaging analyses were augmented to allow in situ analyses of tissue-specific N-glycosylation profiles. In the present study, we outline an improved automated sample preparation method for N-glycan MALDI imaging, which uses in situ PNGase F-mediated release and measurement of N-linked glycans from sections of formalin-fixed murine kidney. The sum of the presented data indicated that N-glycans can be cleaved from proteins within formalin-fixed tissue and characterized using three strategies: (i) extraction and composition analysis through on-target MALDI MS and liquid chromatography coupled to electrospray ionization ion trap MS; (ii) MALDI profiling, where N-glycans are released and measured from large droplet arrays in situ; and (iii) MALDI imaging, which maps the tissue specificity of N-glycans at a higher resolution. Thus, we present a complete, straightforward method that combines MALDI imaging and characterization of tissue-specific N-glycans and complements existing strategies.

The utility of isotope-coded protein labeling for prioritization of proteins found in ovarian cancer patient urine.

Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization.

Investigation of carryover of peptides in nano-liquid chromatography/mass spectrometry using packed and monolithic capillary columns.

This article relates on reversed-phase column technology as the main cause of carryover in the LC-MS/MS analysis of proteomics samples. The separation performance and column carryover was investigated using four capillary columns with different morphologies by monitoring the remaining traces of tryptic peptides of bovine serum albumin in subsequent blank LC-MS runs. The following trend in column carryover was observed: capillary column packed with 3µm porous C18 particles≫2.7µm fused-core C18 packed column>silica C18 monolith≫poly(styrene-co-divinylbenzene) monolith. This is mainly related to the intrinsic properties of the different chromatographic materials, related to surface area and the presence and size of mesopores (stagnant zones where mass transfer is controlled by diffusion). Both isocratic and gradient wash steps with 2-propanol/acetonitrile mixtures were not effective to reduce column carryover. An isocratic wash step using a high acetonitrile percentage or blank gradient reduced carryover with approximately 50%. Nevertheless, it is important to note that effects of column carryover were still observed in a fifth subsequent gradient blank. Although the polymer monolith clearly outperformed the silica materials in terms of carryover, this material exhibited also the lowest loadability, which may be a disadvantage when profiling proteomics mixtures with a broad dynamic range.

Comparison of ZIC-HILIC and graphitized carbon-based analytical approaches combined with exoglycosidase digestions for analysis of glycans from monoclonal antibodies.

Two LC approaches for analysis of therapeutic monoclonal antibodies (MAbs) are presented and compared. In the first approach, zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) of 2-aminobenzamide-labelled glycans was coupled with fluorescence or electrospray ionisation mass spectrometric (ESI-MS) detection. The ZIC-HILIC method enabled relative quantification and identification of major glycan species. The sensitivity of fluorescence detection was higher compared to ESI-MS; however, MS detection enabled identification of co-eluted peaks. The new ZIC-HILIC approach was compared with porous graphitized carbon (PGC) separation of reduced glycans coupled with ESI-MS. Using PGC higher sensitivity was achieved compared to ZIC-HILIC due to the lower chemical background originating from the mobile phase and the derivatisation step, providing detailed information on minor glycan species. Furthermore, PGC exhibited excellent capability for separation of isobaric glycans with various degrees of mannosylation and galactosylation. The structures of glycans from MAbs used in this study were confirmed by exoglycosidase digestions. The two methods were applied to two monoclonal antibodies expressed in Chinese Hamster ovary cell lines and a monoclonal antibody expressed in a murine NS0 cell line. While the fluorescence-based approach is more suitable for routine glycan profiling due to the simplicity of data analysis, MS-based approaches were shown to provide detailed glycosylation analysis of complex glycoprotein samples.

Analysis of phosphopeptide changes as spermatozoa acquire functional competence in the epididymis demonstrates changes in the post-translational modification of Izumo1.

Spermatozoa are functionally inert when they emerge from the testes. Functional competence is conferred upon these cells during a post-testicular phase of sperm maturation in the epididymis. Remarkably, this functional transformation of epididymal spermatozoa occurs in the absence of nuclear gene transcription or protein translation. To understand the cellular mechanisms underpinning epididymal maturation, we have performed a label-free, MS-based, comparative quantification of peptides from caput, corpus and caudal epididymal spermatozoa. In total, 68 phosphopeptide changes could be detected during epididymal maturation corresponding to the identification of 22 modified proteins. Included in this list are the sodium-bicarbonate cotransporter, the sperm specific serine kinase 1, AKAP4 and protein kinase A regulatory subunit. Furthermore, four phosphopeptide changes came from Izumo1, the sperm-egg fusion protein, in the cytoplasmic segment of the protein. 2D-PAGE confirmed that Izumo1 is post-translationally modified during epididymal transit. Interestingly, phosphorylation on Izumo1 was detected on residue S339 in the caput and corpus but not caudal cells. Furthermore, Izumo1 exhibited four phosphorylated residues when spermatozoa reached the cauda, which were absent from caput cells. A model is advanced suggesting that these phospho-regulations are likely to act as a scaffold for the association of adaptor proteins with Izumo1 as these cells prepare for fertilization.

LDI and ESI MS as well as low energy CID of a self-assembling nanorod-forming fullerene derivative.

An amphiphatic fullerene derivative (8-(N-Methyl-Fullero-Pyrrolidinium-1-yl-chloride)-3,6-Dioxaoctan-1-Ammonium Chloride (MFPDAC)), which is of great interest in nanotechnology due to the fact that it forms self-assembling fullerenic nanorods, has been structurally characterized with emphasis to its purity and thermal treatment of a formed nanorod film (on a LDI target) by means of laser desorption/ionization (LDI) coupled with high-resolution curved field reflectron time-of-flight (TOF) mass spectrometry, and by low energy MS/MS as well as in-source fragmentation experiments applying an quadrupole ion trap (QIT) combined with a two-stage reflectron TOF analyzer. The interpretation of LDI results has been supplemented by ESI QIT MS(n) (n = 1-3), as well as high-resolution ESI reflectron TOF mass spectrometric experiments. Based on the experimental data obtained by both desorption/ionization techniques, various types of analyzers and sample treatments, we could completely characterize MFPDAC and further found out that the investigated sample was not entirely free of impurities. Furthermore, the envisaged loss of the derivative sidechain upon the heat treatment in vacuum of the self-assembled nanorod sample film on a metallic substrate could be successfully monitored by LDI MS.

Zwitterionic-type hydrophilic interaction nano-liquid chromatography of complex and high mannose glycans coupled with electrospray ionisation high resolution time of flight mass spectrometry.

In this study we describe a new method for rapid and sensitive analysis of reduced high mannose and complex glycans using zwitterionic-type hydrophilic interaction nano-liquid chromatography (nano ZIC-HILIC, 75 µm I.D.×150 mm) coupled with high resolution nanoelectrospray ionisation time of flight mass spectrometry (nano ESI-TOF-MS). The retention of neutral glycans increases with increasing molecular weight and is higher for high mannose glycans than for complex-type glycans. The selectivity of ZIC-HILIC for sialylated glycans differs from that for the neutral glycans and is believed to involve electrostatic repulsion; therefore, charged glycans are eluted earlier than neutral glycans with comparable molecular weight. Due to the improved sensitivity achieved by employing a ZIC-HILIC nano-column, a range of less common complex glycans has been studied and the high resolution mass spectrometry enabled confirmation of glycan composition for the proposed structures. Good sensitivity for glycans was achieved without prior fluorescent labelling, and the time of the analysis was significantly reduced compared to the separation of glycans on a conventional-size column. The proposed method offers a fast and sensitive approach for glycan profiling applied to analysis of biopharmaceuticals.

An optimized method for the determination of perfluorooctanoic acid, perfluorooctane sulfonate and other perfluorochemicals in different matrices using liquid chromatography/ion-trap mass spectrometry.

Perfluorochemicals (PFC's) are widely spread in the environment and have been detected in blood of wildlife and humans world-wide. Recently, various toxic effects of PFC's in laboratory rats have been demonstrated, resulting in increased government concerns regarding the presence of PFC's in the environment and the implications they have on human health. In the last decade, various analytical methods have been developed for the analysis of PFC's in different matrices whereby the majority of methods have utilised liquid chromatography coupled with mass spectrometry (LC-MS). Here we describe an optimized method for the quantitation of PFC's, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), in food packaging, polytetrafluoroethylene (PTFE) sealant tape and drinking water. The method involved PFC's extraction via off-line SPE followed by separation using reversed-phase liquid chromatography on a Phenyl-Hexyl column coupled with ion-trap (IT) mass spectrometric detection. The optimized approach minimized ion-suppression effects commonly seen with conventional elution buffers, improving detection limits down to 25 pg/mL and allowed effective quantitation down to 50 pg/mL for PFOA and PFOS. The optimized LC-MS method detected PFOA and other PFC's in microwave popcorn packaging and PFOA in PTFE sealant tape in the low µg/kg. In all samples, PFOS was not detected.

High resolution TOF MS coupled to CE for the analysis of isotopically resolved intact proteins.

Intact protein analysis by mass spectrometry is of great interest for the characterisation of biotechnological products. Exact mass measurement in combination with isotopic resolution allows the detection of modifications leading to small mass changes like deamidation or reduction of disulfide bonds directly on the level of the intact protein. Here, a concept is presented based on time-of-flight mass spectrometry. A bench top TOF MS and a high resolution TOF MS were used to resolve the isotopes of intact recombinant human growth hormone and intact human erythropoietin, respectively. Thus, these 22 and around 30kDa large proteins can be characterised sensitively in great detail and along with capillary electrophoretic separation unambiguous identification of minor protein modifications like deamidation is possible.

Use of titanium dioxide to find phosphopeptide and total protein changes during epididymal sperm maturation.

Although the overall performance of modern mass spectrometers has increased, proteomic analysis of complex samples still requires prefractionation either at the protein or peptide level to allow for in-depth analysis of normal cellular function. Here, we report a novel way to identify protein changes occurring during sperm development through the epididymis. Phosphopeptides were first enriched from either the rat caput or caudal regions of the epididymides using TiO(2), and the profiles then quantitatively compared. We show that 77 TiO(2)-enriched peptides become significantly modified in the epididymis, equating to 53 proteins. Through the use of immunoblot analysis, we confirmed that three proteins, ornithine-decarboxylase antizyme 3, heat-shock protein 90α, and testis-lipid binding protein, undergo major protein loss during epididymal passage. Many other proteins, including t-complex protein 10 and Spata18 show testis unique expression, appear to undergo phosphorylation during this same time frame. These data provide mechanistic insight into the means by which spermatozoa acquire functionality during epididymal transit.

Capillary zone electrophoresis-mass spectrometry for the characterization of isoforms of intact glycoproteins.

Electrospray ionization-mass spectrometry (ESI-MS) is a powerful tool for the characterization of intact proteins. However, complex samples require separation in order to obtain clear mass spectra and avoid matrix interaction; capillary electrophoresis (CE) has been shown to be a powerful separation technique for intact proteins. Herein, we present a method based on capillary zone electrophoretic (CZE) separation coupled online with mass spectrometric protein detection. While this approach is suitable for the separation and characterization of various intact proteins, the emphasis is placed on the separation of glycoforms of various and rather complex glycoproteins. The method has been successfully applied to the analysis of glycoproteins, e.g., erythropoietin, fetuin, and alpha-acid glycoprotein.

Fast capillary electrophoresis coupled with time-of-flight mass spectrometry under separation conditions of high electrical field strengths.

An experimental approach is presented that enables very fast capillary electrophoretic separations in conjunction with time-of-flight mass spectrometry. Field strengths exceeding 1 kV cm(-1) have been applied for separations of model analytes resulting in migration times on the timescale of seconds.