Nitrile imines produced by photodissociation of 2,5-diaryltetrazoles undergo cross-linking reactions with amide groups in peptide-tetrazole (tet-peptide) conjugates and a tet-peptide-dinucleotide complex. Tetrazole photodissociation in gas-phase ions is efficient, achieving ca. 50% conversion with 2 laser pulses at 250 nm. The formation of cross-links was detected by CID-MS3 that showed structure-significant dissociations by loss of side-chain groups and internal peptide segments. The structure and composition of cross-linking products were established by a combination of UV-vis action spectroscopy and cyclic ion mobility mass spectrometry (c-IMS). The experimental absorption bands were found to match the bands calculated for vibronic absorption spectra of nitrile imines and cross-linked hydrazone isomers. The calculated collision cross sections (CCSth) for these ions were related to the matching experimental CCSexp from multipass c-IMS measurements. Loss of N2 from tet-peptide conjugates was calculated to be a mildly endothermic reaction with ΔH0 = 80 kJ mol-1 in the gas phase. The excess energy in the photolytically formed nitrile imine is thought to drive endothermic proton transfer, followed by exothermic cyclization to a sterically accessible peptide amide group. The exothermic nitrile imine reaction with peptide amides is promoted by proton transfer and may involve an initial [3 + 2] cycloaddition followed by cleavage of the oxadiazole intermediate. Nucleophilic groups, such as cysteine thiol, did not compete with the amide cyclization. Nitrile imine cross-linking to 2'-deoxycytidylguanosine was found to be >80% efficient and highly specific in targeting guanine. The further potential for exploring nitrile-imine cross-linking for biomolecular structure analysis is discussed.
The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein. SIGNIFICANCE: Even though the amine reactive BS2G and DSG cross-linkers have the same length of spacer and are based on N-hydroxysuccinimidic group, our data showed that each of them formed preferentially different cross-links. We demonstrated that the choice of cross-linker can have a significant impact on the output data for structural characterization of biomolecules. Using equimolar mixtures of DSG with d6-BS2G, and BS2G with d6-DSG, we established that the polar BS2G preferentially bound to polar regions of modified molecule, whereas non-polar DSG bound to hydrophobic regions. This phenomenon established that the mixture of polar and non-polar cross-linkers acted as an efficient tool for the determination of distance constraints in proteins.
Protein cross-linking has assumed an irreplaceable role in structural proteomics. Recently, significant efforts have been made to develop novel mass spectrometry (MS)-cleavable reagents. At present, only water-insoluble MS-cleavable cross-linkers are commercially available. However, to comprehensively analyse the various chemical and structural motifs making up proteins, it is necessary to target different protein sites with varying degrees of hydrophilicity. Here we introduce the new MS-cleavable cross-linker disulfodisuccinimidyl dibutyric urea (DSSBU), which we have developed in-house for this purpose. DSSBU contains an N-hydroxysulfosuccinimide (sulfo-NHS) reactive group, so it can serve as a water-soluble counterpart to the widely used cross-linker disuccinimidyl dibutyric urea (DSBU). To investigate the applicability of DSSBU, we compared the efficacy of four similar cross-linkers: bis[sulfosuccinimidyl] suberate (BS3), disuccinimidyl suberate (DSS), DSBU and DSSBU with bovine serum albumin. In addition, we compared the efficacy of DSBU and DSSBU with human haemoglobin. Our results demonstrate that the sulfo-NHS group ensures the superior water solubility of DSSBU and thus negates the need for organic solvents such as dimethyl sulfoxide while preserving the effectivity of urea-based MS-cleavable crosslinkers such as DSBU. Additionally, it makes it possible to target polar regions in proteins. The data gathered are available via ProteomeXchange under identifier PXD055284. SIGNIFICANCE: We have synthesized the novel protein cross-linker DSSBU, which combines sulfo-NHS ester chemistry with a mass spectrometry-cleavable urea group. This makes DSSBU a water-soluble, MS-cleavable cross-linker that reacts with amino groups. To our knowledge, it is the first cross-linker which combines all three of these characteristics. We have tested the performance of our novel cross-linker on bovine serum albumin, a model widely used by the cross-linking mass spectrometry community, and on human haemoglobin. We have comprehensively assessed the performance of DSSBU and compared its efficacy with that of three other cross-linkers in current use (BS3, DSS and DSBU). We conclude that our novel cross-linker surpasses its MS-non-cleavable analogue BS3 in performance and that its water solubility eliminates the need for organic solvents while its hydrophilicity allows for the targetting of polar regions in proteins. Therefore, it will likely become a significant addition to the portfolio of N-hydroxysuccinimide ester cross-linkers.
- MeSH
- Mass Spectrometry methods MeSH
- Humans MeSH
- Urea chemistry MeSH
- Proteomics methods MeSH
- Cross-Linking Reagents * chemistry MeSH
- Serum Albumin, Bovine chemistry MeSH
- Cattle MeSH
- Succinimides * chemistry MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
We synthesized seven different polymethacrylate monolithic capillary columns using N,N-dimethyl-N-metacryloxyethyl-N-(3-sulfopropyl) ammonium betaine functional monomer (MEDSA) and various cross-linking monomers differing in the polarity and size. The efficiency of monolithic columns for polar low-molecular compounds in the aqueous normal-phase (HILIC) mode depends rather on the polarity, than on the size of the cross-linker molecules. Cross-linking molecules, which exhibit high polarity can produce poly(methacrylate) monoliths with an increase in pore sizes below 50nm. Columns prepared with pentaerythritol triacrylate or bisphenol A dimethacrylate cross-linkers show large inner pore (mesopore) porosity, and provide poor efficiency, whereas columns with trioxyethylene dimethacrylate (TriEDMA) and tetraoxyethylene dimethacrylate (TeEDMA) cross-linkers showed poor permeability. Columns prepared using dioxyethylene dimethacrylate (DiEDMA), and especially glycerolate dimethacrylate (BIGDMA) cross-linkers, showed best efficiency, with more 60,000-70,000 theoretical plates/m, almost twice in comparison to (poly)methylene dimethacrylate (HEDMA) in the HILIC mode. All columns show dual retention mechanism and can be used for separations of low-molcular compounds such as phenolic acids in the HILIC mode in acetonitrile-rich mobile phases and in the reversed-phase mode in mobile phases with higher concentrations of water. Some columns show broad pore distribution and can be used for size-exclusion chromatography of non-polar polymers in tetrahydrofuran.
We synthesized 8 polymethacrylate monolithic capillary columns using laurylmethacrylate functional monomer and various cross-linking monomers differing in the polarity and size. The efficiency of monolithic columns for low-molecular compounds significantly improved with increasing number of repeat non-polar methylene groups in the cross-linker molecules, correlating with greater proportion of small pores with size less than 50 nm. The best efficiency with HETP=25 μm for alkylbenzenes was achieved for columns prepared using hexamethylene dimethacrylate (HEDMA). Columns prepared with polar (poly)oxyethylene dimethacrylate cross-linkers show also improved efficiency with increasing chain length and generally better performance in comparison to the (poly)methylene dimethacrylate cross-linkers of comparable size, however with less apparent effects of the chain lengths on the pore distribution. The monolithic columns prepared with tetraoxyethylene dimethacrylate (TeEDMA) showed the best efficiency of all the columns tested, corresponding to HETP=15 μm (approx. 70,000 theoretical plates/m), show excellent column-to-column reproducibility with standard deviations of 2.5% in retention times, good permeability and low mass transfer resistance, so that is suitable for fast separation of low-molecular compounds in 2 min or less. By modification of the fused-silica capillary inner walls pre-treatment procedure, very good long-term stability was achieved even in 0.5 mm i.d. capillary format. The TeEDMA column can be also used for size-exclusion chromatography of lower non-polar synthetic polymers, whereas it is less suitable for separations of proteins than the HEDMA column.
- MeSH
- Benzene Derivatives isolation & purification MeSH
- Chromatography, Reverse-Phase instrumentation MeSH
- Polymethacrylic Acids chemistry MeSH
- Polymerization MeSH
- Porosity MeSH
- Proteins isolation & purification MeSH
- Cross-Linking Reagents chemistry MeSH
- Reproducibility of Results MeSH
- Cattle MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca(2+) response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)(2) or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcεRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)(2) fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcεRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.
- MeSH
- Adaptor Proteins, Signal Transducing metabolism MeSH
- Tetraspanin 29 physiology MeSH
- Fusion Regulatory Protein 1, Light Chains metabolism MeSH
- Antigens metabolism MeSH
- Models, Biological MeSH
- Cell Membrane metabolism MeSH
- Chemotaxis MeSH
- Cytoskeleton metabolism MeSH
- Phosphoproteins metabolism MeSH
- Phosphorylation MeSH
- Glucuronidase metabolism MeSH
- Immunoglobulin Fab Fragments chemistry MeSH
- Rats MeSH
- Mast Cells cytology MeSH
- Membrane Proteins metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Rats, Wistar MeSH
- Receptors, IgE metabolism MeSH
- Amino Acid Transport System y+ metabolism MeSH
- Tyrosine chemistry MeSH
- Calcium metabolism MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In order to learn more about the molecular basis for the inhibition of DNA replication produced by antitumor platinum drugs, we investigated DNA polymerization using DNA templates site-specifically modified with the 1,2-GG intrastrand cross-link of dinuclear bifunctional [{trans-PtCl(NH(3))(2)}(2){l-spermidine-N1,N8}](3+)(BBR3571) or conventional mononuclear cisplatin. These cross-links which have the same nature, but differ in the size and character of the conformational alteration induced in double-helical DNA, were analyzed for bypass ability with reverse transcriptase of human immunodeficiency virus type 1 and Klenow fragment of DNA polymerase I deficient in exonuclease activity. We found that the 1,2-GG intrastrand CL of BBR3571 inhibited DNA translesion synthesis markedly more than the same adduct of cisplatin. This result was explained by a larger size of the cross-link of BBR3571 and by a flexibility induced in DNA by this cross-link which can make the productive binding of this adduct at the polymerase site more difficult.
Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.
- MeSH
- CD48 Antigen metabolism MeSH
- CD59 Antigens metabolism MeSH
- Immunoglobulin Fab Fragments chemistry immunology metabolism MeSH
- Jurkat Cells MeSH
- Humans MeSH
- Liposomes chemistry MeSH
- Lymphoma immunology metabolism pathology MeSH
- Antibodies, Monoclonal chemistry immunology metabolism MeSH
- Mice MeSH
- Tumor Cells, Cultured MeSH
- Peptide Fragments immunology metabolism MeSH
- Pulmonary Surfactant-Associated Protein D immunology metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
A number of biologically important molecules, such as DNA, proteins, and antibodies, are routinely conjugated with fluorescent tags for high-sensitivity analyses. Here, the application of quantum dots in the place of bright and size-tunable luminophores is studied. Several selected bioconjugation reactions via zero-length cross-linkers, long-chain linkers, and oriented methods for linking of quantum dots with proteins were tested. Anti-ovalbumin, anti-proliferating cell nuclear antigen, anti-hemagglutinin, and anti-CD3 membrane protein as model antibodies and annexin V were used as high-specificity selectors. The reaction yield and efficiency of the prepared immunoluminescent probes were tested by capillary zone electrophoresis with laser-induced fluorescence detection.
- MeSH
- Electrophoresis, Capillary MeSH
- Immunoassay instrumentation methods MeSH
- Quantum Dots MeSH
- Luminescent Measurements instrumentation methods MeSH
- Molecular Probes chemistry MeSH
- Proteins analysis isolation & purification MeSH
- Antibodies chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Intermediate filaments (IFs) are essential constituents of the metazoan cytoskeleton. A vast family of cytoplasmic IF proteins are capable of self-assembly from soluble tetrameric species into typical 10-12 nm wide filaments. The primary structure of these proteins includes the signature central 'rod' domain of ~ 300 residues which forms a dimeric α-helical coiled coil composed of three segments (coil1A, coil1B and coil2) interconnected by non-helical, flexible linkers (L1 and L12). The rod is flanked by flexible terminal head and tail domains. At present, the molecular architecture of mature IFs is only poorly known, limiting our capacity to rationalize the effect of numerous disease-related mutations found in IF proteins. Here we addressed the molecular structure of soluble vimentin tetramers which are formed by two antiparallel, staggered dimers with coil1B domains aligned (A11 tetramers). By examining a series of progressive truncations, we show that the presence of the coil1A domain is essential for the tetramer formation. In addition, we employed a novel chemical cross-linking pipeline including isotope labelling to identify intra- and interdimeric cross-links within the tetramer. We conclude that the tetramer is synergistically stabilized by the interactions of the aligned coil1B domains, the interactions between coil1A and the N-terminal portion of coil2, and the electrostatic attraction between the oppositely charged head and rod domains. Our cross-linking data indicate that, starting with a straight A11 tetramer, flexibility of linkers L1 and L12 enables 'backfolding' of both the coil1A and coil2 domains onto the tetrameric core formed by the coil1B domains. Through additional small-angle X-ray scattering experiments we show that the elongated A11 tetramers dominate in low ionic strength solutions, while there is also a significant structural flexibility especially in the terminal domains.
