Vapor conjugation of toluene diisocyanate to specific lysines of human albumin

Exposure to toluene diisocyanate (TDI), an industrially important crosslinking agent used in the production of polyurethane products, can cause asthma in sensitive workers. Albumin has been identified as a major reaction target for TDI in vivo, and TDI-albumin reaction products have been proposed to serve as exposure biomarkers and to act as asthmagens, yet they remain incompletely characterized. In the current study, we used a multiplexed tandem mass spectrometry (MS/MS) approach to identify the sites of albumin conjugation by TDI vapors, modeling the air/liquid interface of the lung. Vapor phase TDI was found to react with human albumin in a dose-dependent manner, with up to 18 potential sites of conjugation, the most susceptible being Lys351 and the dilysine site Lys413-414. Sites of vapor TDI conjugation to albumin were quantitatively limited compared with those recently described for liquid phase TDI, especially in domains IIA and IIIB of albumin. We hypothesize that the orientation of albumin at the air/liquid interface plays an important role in vapor TDI conjugation and, thus, could influence biological responses to exposure and the development of in vitro assays for exposure and immune sensitivity.     

Determination of a new biomarker in subjects exposed to 4,4′-methylenediphenyl diisocyanate

4,4′-Methylenediphenyl diisocyanate (MDI) is the most important of the isocyanates used as intermediates in the chemical industry. Among the main types of damage after exposure to low levels of MDI are lung sensitization and asthma. Albumin adducts of MDI might be involved in the etiology of sensitization reactions. This work presents a liquid chromatography (LC)–mass spectrometry (MS/MS) procedure for determination of isocyanate-specific albumin adducts in humans. MDI formed adducts with lysine of albumin: MDI–Lys and AcMDI–Lys. The MDI–Lys levels, 25th, 50th, 75th, 90th percentile, were 0, 65.2, 134, 244?fmol mg^?1 and 0, 30.5, 57.4, 95.8?fmol mg^?1 in the exposed construction and factory workers, respectively. This new biomonitoring procedure will allow assessment of suspected exposure sources and may contribute to the identification of individuals who are particularly vulnerable for developing bronchial asthma and other respiratory diseases after exposure to isocyanates.     

Determination of the toluene diisocyanate binding sites on human serum albumin by tandem mass spectrometry

Diisocyanates are highly reactive chemical compounds widely used in the manufacture of polyurethanes. Although diisocyanates have been identified as causative agents of allergic respiratory diseases, the specific mechanism by which these diseases occur is largely unknown. To better understand the chemical species produced when diisocyanates react with protein, tandem mass spectrometry was employed to unambiguously identify the binding sites of the industrially important isomers, 2,4- and 2,6-toluene diisocyanate, on human serum albumin at varying diisocyanate/protein ratios. The 2,4-isomer results in approximately 2-fold higher conjugation product ion abundances than does the 2,6-isomer, suggesting that the 2,4-isomer has a higher reactivity toward albumin. Both isomers preferentially react with the N-terminal amine of the protein and the ε-NH₂ of lysine. At a low (1:2) diisocyanate/protein ratio, five binding sites are identified, whereas at a high (40:1) ratio, near-stoichiometric conjugation is observed with a maximum of 37 binding sites identified. Binding sites observed at the lowest conjugation ratios are conserved at higher binding ratios, suggesting a subset of 5–10 preferential binding sites on albumin. Diisocyanate–protein conjugation results in a variety of reaction products, including intra- and intermolecular crosslinking, diisocyanate self-polymerization, and diisocyanate hydrolysis.     

A rapid approach for characterization of thiol-conjugated antibody–drug conjugates and calculation of drug–antibody ratio by liquid chromatography mass spectrometry

We present the demonstration of a rapid “middle-up” liquid chromatography mass spectrometry (LC–MS)-based workflow for use in the characterization of thiol-conjugated maleimidocaproyl-monomethyl auristatin F (mcMMAF) and valine-citrulline-monomethyl auristatin E (vcMMAE) antibody–drug conjugates. Deconvoluted spectra were generated following a combination of deglycosylation, IdeS (immunoglobulin-degrading enzyme from Streptococcus pyogenes) digestion, and reduction steps that provide a visual representation of the product for rapid lot-to-lot comparison—a means to quickly assess the integrity of the antibody structure and the applied conjugation chemistry by mass. The relative abundance of the detected ions also offer information regarding differences in drug conjugation levels between samples, and the average drug–antibody ratio can be calculated. The approach requires little material (<100 μg) and, thus, is amenable to small-scale process development testing or as an early component of a complete characterization project facilitating informed decision making regarding which aspects of a molecule might need to be examined in more detail by orthogonal methodologies.     

Simple, direct conjugation of bacterial O-SP-core antigens to proteins: development of cholera conjugate vaccines

Bacterial O-SP-core antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. Introduction of spacers (linkers) to either O-SP-core antigens or protein carriers, which is involved in commonly applied protocols, is not required. The newly developed method described here consists of preparation of a squaric acid monoester derivative of O-SP-core antigen, utilizing the amino group inherent in the core, and reaction of the monoester with the carrier protein. The intermediate monoester can be easily purified; its conjugation can be monitored by SELDI-TOF mass spectrometry and, thus, readily controlled, since the conjugation can be terminated when the desired carbohydrate-protein ratio is reached. Here, we describe production of conjugates containing the O-SP-core antigen of Vibrio cholerae O1, the major cause of cholera, a severe dehydrating diarrheal disease of humans. The resultant products are recognized by convalescent phase sera from patients recovering from cholera in Bangladesh, and anti-O-SP-core-protein responses correlate with plasma antilipopolysaccharide and vibriocidal responses, which are the primary markers of protection from cholera. The results suggest that such conjugates have potential as vaccines for cholera and other bacterial diseases.     

Characterization of morphine-glucose-6-phosphate dehydrogenase conjugates by mass spectrometry

A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. To improve our understanding of the antibody-induced inhibition mechanism, we characterized morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine 3-glucuronide NHS ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods, including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially the cause of loss of activity due to the coupling reaction.     

Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin

Protein haptenation by polyurethane industrial intermediate 4,4′-methylene diphenyl diisocyanate (MDI) is thought to be an important step in the development of diisocyanate (dNCO)-specific allergic sensitization; however, MDI-haptenated albumins used to screen specific antibody are often poorly characterized. Recently, the need to develop standardized immunoassays using a consistent, well-characterized dNCO-haptenated protein to screen for the presence of MDI-specific IgE and IgG from workers’ sera has been emphasized and recognized. This has been challenging to achieve due to the bivalent electrophilic nature of dNCOs, leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the current study, MDI was reacted with human serum albumin (HSA) and hemoglobin (Hb) at molar ratios ranging from 1:1 to 40:1 MDI/protein. Adducts were characterized by (i) loss of available 2,4,6-trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (ii) electrophoretic migration in polyacrylamide gels, (iii) quantification of methylene diphenyl diamine following acid hydrolysis, and (iv) immunoassay. Concentration-dependent changes in all of the above noted parameters were observed, demonstrating increases in both number and complexity of conjugates formed with increasing MDI concentrations. In conclusion, a series of bioanalytical assays should be performed to standardize MDI–antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers that in total indicate degree of intra- and intermolecular cross-linking, number of dNCOs bound, number of different specific binding sites on the protein, and degree of immunoreactivity.     

Synthesis of fluorescent analogs of alpha-conotoxin MII

Alpha-conotoxins (alpha-CTxs) are small peptides that are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) and have been used to study the kinetics of nAChRs. Alpha-CTx MII, from the venom of Conus magus, has been shown to potently block both rat alpha3beta2 and rat chimeric alpha6/alpha3beta2beta3 cloned nAChRs expressed in Xenopus oocytes. Tetramethylrhodamine (TMR), Bodipy FL, Alexa Fluor 488, and terbium chelates (TbCh) are fluorescent molecules that can be reacted with the N-terminus of the conopeptide to produce fluorescent conjugates. TMR and Bodipy FL were individually conjugated to alpha-CTx MII using different succinimidyl ester amine labeling reactions resulting in the formation of carboxamide conjugates. Alexa Fluor 488 succinimidyl ester conjugation reaction yielded low amounts of conjugate. TbCh was also individually reacted with the N-terminus of MII using the isothiocyanate conjugation reaction resulting in the formation of a thiourea conjugate. The conjugates were purified using reverse-phase high-pressure liquid chromatography (RP-HPLC) and their masses verified by matrix-assisted laser desorption-ionization with time-of-flight mass spectroscopy (MALDI-TOF MS). When tested on target nAChRs expressed in Xenopus oocytes, TMR-MII, Bodipy FL-MII, and TbCh-MII potently blocked the response to acetylcholine with slow off-rate kinetics. These fluorescent conjugates can be used to localize specific subtypes of neuronal nAChRs or ligand-binding sites within receptors in various tissue preparations; additionally, they may also be used to study conformational changes in receptors using fluorescence or lanthanide-based resonance energy transfer.     

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

Antibody–drug conjugates (ADCs) are antibody‐based therapeutics that have proven to be highly effective cancer treatment platforms. They are composed of monoclonal antibodies conjugated with highly potent drugs via chemical linkers. Compared to cysteine‐targeted chemistries, conjugation at native lysine residues can lead to a higher degree of structural heterogeneity, and thus it is important to evaluate the impact of conjugation on antibody conformation. Here, we present a workflow involving native ion mobility (IM)‐MS and gas‐phase unfolding for the structural characterization of lysine‐linked monoclonal antibody (mAb)–biotin conjugates. Following the determination of conjugation states via denaturing Liquid Chromatography‐Mass Spectrometry (LC–MS) measurements, we performed both size exclusion chromatography (SEC) and native IM‐MS measurements in order to compare the structures of biotinylated and unmodified IgG1 molecules. Hydrodynamic radii (Rh) and collision cross‐sectional (CCS) values were insufficient to distinguish the conformational changes in these antibody–biotin conjugates owing to their flexible structures and limited instrument resolution. In contrast, collision induced unfolding (CIU) analyses were able to detect subtle structural and stability differences in the mAb upon biotin conjugation, exhibiting a sensitivity to mAb conjugation that exceeds native MS analysis alone. Destabilization of mAb–biotin conjugates was detected by both CIU and differential scanning calorimetry (DSC) data, suggesting a previously unknown correlation between the two measurement tools. We conclude by discussing the impact of IM‐MS and CIU technologies on the future of ADC development pipelines.     

Direct protein–protein conjugation by genetically introducing bioorthogonal functional groups into proteins

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein–protein conjugates has gained research interest. In this study, site-specific protein–protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein–protein conjugates and protein complexes of biochemical significance.     

Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry

Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.     

Light‐microscopic examinations of sperm in several lineages of Carabidae (Insecta: Coleoptera): Implications for the evolution of sperm conjugation

Sperm show marked morphological diversity, but the processes and mechanisms driving this diversity have not been fully elucidated. The beetle family Carabidae represents a potential model system for studying sperm trait evolution. In this study, sperm traits (mainly conjugation and sperm conjugate gross morphology) of 42 species from nine subfamilies of Carabidae were examined using light microscopy. Except in Harpalinae, the type of conjugation was shared by all members of a particular subfamily: in Carabinae, Elaphrinae, Patrobinae and Brachinae, sperm conjugates were observed in which variable numbers of sperm clumped together; in Nebriinae, Cicindelinae and Trechinae, sperm were not organized as conjugates but were present individually; and in Broscinae, both individual sperm and sperm conjugates were observed. In the remaining subfamily, Harpalinae, sperm conjugates were formed in most species, but a loss of conjugation was observed in some species. Mapping the observed sperm traits onto within‐family molecular phylogenetic trees suggested that sperm conjugation was ancestral, with loss of conjugation evolving in several lineages. In sperm conjugates, a short spermatostyle (the axis of sperm conjugates) was the ancestral state, while a long spermatostyle evolved in subsequent lineages. In the long spermatostyle trait, the flexible type without a conspicuous 3D structure was ancestral, while the type with a conspicuous 3D structure, such as the spiral structure, evolved in derived lineages.     

Bisphosphonate conjugation to proteins as a means to impart bone affinity

Growth factors are endogenous proteins capable of stimulating new bone formation, but their clinical benefit for systemic stimulation of bone mass has not been demonstrated. The critical challenge is to deliver a significant dose of the proteins to bone after intravenous injection. This challenge may be overcome by derivatizing proteins with ligands that exhibit a high bone affinity (e.g., bisphosphonates). To demonstrate the feasibility of this approach, 1-amino-1,1-diphosphonate methane (aminoBP) was conjugated to a model protein, albumin. The conjugation was performed by (1) converting the amino group of aminoBP to a thiol group using 2-iminothiolane, (2) derivatizing the albumin amino groups with a thiol-reactive sulfosuccinimidyl-4-(N-maleimidomethyl)-1-cyclohexane carboxylate, and (3) reacting the derivatized albumin with thiolated aminoBP. Typically, 1-4 aminoBP molecules per albumin were obtained. The conjugated albumin exhibited a high affinity to hydroxyapatite that was proportional to the extent of conjugation. The conjugates were shown to exhibit a high affinity to bone matrix in vitro in a serum-containing medium. Once bound to bone matrix, the conjugates were found to desorb more slowly than the unmodified albumin, especially from bone whose organic matrix was removed by ashing. In conclusion, conjugation of bisphosphonates to albumin was shown to impart a high bone affinity to the protein, and such conjugates can be potentially targeted to bone.     

Determination of estrogens by radioimmunoassay with antibodies to estrogen-C6-conjugates. I. Synthesis of estrone-, estradiol-17 -, and estriol-6-albumin conjugates

6-oxoestriol 6-carboxymethoxime, 6-oxoestradiol-17beta 6-carboxymethoxime, and 6-oxoestrone 6-carboxymethoxime were prepared and each was coupled to bovine serum albumin by means of the mixed anhydride technique. Standard methods were used to characterize the reaction products and intermediates. The results indicate that the estrone-, estradiol-17beta-, and estriol-6-albumin conjugates, as prepared in this study, can be used as antigens which induce specific antibodies to the corresponding haptens.     

Products of spontaneous conjugation of aflatoxins with bovine serum albumin: immunochemical properties

Products of spontaneous conjugation of aflatoxins B1, G1, and G2 with bovine serum albumin (BSA) were shown to interact with antibodies against aflatoxins. Solid-phase BSA conjugates inhibited the binding of aflatoxins by anti-aflatoxin antibodies. Antisera against BSA-B1, BSA-G1, and BSA-G2 were obtained and their specificity, determined. The mechanisms of spontaneous binding of aflatoxins by proteins are discussed.     

Radioimmunoassays for trans-zeatin and related cytokinins

Radioimmunoassays for the quantitation of trans-zeatin and related cytokinins have been developed. Antisera produced against bovine serum albumin conjugates of trans-zeatinriboside have a high affinity (K_a=2.4.10^-11 M) for zeatinriboside and for zeatin, but show a negligible cross reaction to isopentenyladenosine (0.1%) and cis-zeatinriboside (0.4%), only a slight cross reaction to dihydrozeatin (1.7%), and no cross reaction at all to other purines, such as adenosine and related, compounds, was observed. The assays are sensitive and measuring ranges extend from 0.06–30 pmol (0.02–10 ng) of zeatinriboside. This has been achieved by employing as tracers immunoreactive zeatin derivatives with high-specific activity, (tritiated zeatinriboside-dialcohol: 8.37.10¹¹ Bq mmol^-1 and zeatinribosyl-[¹²⁵I]tyramine: ca. 1.9.10¹³ Bq mmol^-1. The detection limit is 40 fmol (15pg) for the assay employing the tritiated tracer, and assay reproducibility is high (variation coefficients of triplicates less than 5%). Several hundred assays can be completed in one day, and, due to the high specificity of this assay, crude extracts may be used for analysis. The course of zeatin levels in developing fruits of Lycopersicon esculentum cv. Moneymaker is given.Abbreviations GC gas chromatography - LC liquid chromatography - MS mass spectroscopy - RIA radioimmunoassay - TLC thin layer chromatography - UB unspecific binding Part 12 in the Series: Use of Immunoassay in Plant Science     

Assessing protein-RNA interactions using microfluidic capillary mobility shift assays

The dynamic range of plasma protein abundance, ranging from milligrams to picograms per milliliter, makes characterization of this proteome nearly impossible with current analytical methods. Plasma preprocessing by high-abundance protein depletion may concomitantly remove important diagnostic information. This article describes an original chromatographic procedure to isolate proteins bound to human serum albumin (HSA). Using HSA as an “affinity agent”, we significantly improved the detection and identification of HSA ligands by two-dimensional liquid chromatography tandem mass spectrometry (2D LC–MS/MS). Some of the characterized species were not previously reported in published blood databases. Albumin-binding proteins may be classified as belonging to several putative functional categories and span a wide variety of predicted physiological functions.     

Emergence and properties of spice and bath salts: A medicinal chemistry perspective

Over the past five years the number of internet sites advertising “legal highs” has literally exploded, as have user reports of experiences (both pleasurable and frightening) with these substances and the number of emergency room visits by users. Although the majority of these “legal highs” have been described as bath salts and herbal extracts, most contain neither plant derived compounds nor components of personal hygiene products. So-called “bath salts” largely contain synthetic analogs of the natural compound Khat; spice-related materials, claimed to be “legal marijuana,” are mostly synthetic analogs of cannabinoid receptor ligands that were developed as research tools. This review describes the emergence and properties of these two groups of “legal highs” from a medicinal chemist's perspective.     

Synthesis, isolation, and characterization of conjugates of ovalbumin with monomethoxypolyethylene glycol using cyanuric chloride as the coupling agent

The experimental conditions for the preparation of conjugates of ovalbumin (OA) and monomethoxypolyethylene glycol (mPEG) of a preselected average degree of conjugation, n, using cyanuric chloride as the coupling agent, have been investigated with emphasis on purification and characterization of the products. These conjugates served as prototypes of tolerogenic mPEG derivatives of antigenic proteins which were capable of suppressing in mammals the immunological response to the corresponding unmodified antigens. In other studies in this laboratory, the tolerogenicity of OA(mPEG)n conjugates was found to be a function of n. The reproducibility of the reaction leading to the production of OA(mPEG)n conjugates was shown to depend primarily on the reactivity of the mPEG-cyanuric chloride intermediate, which--for best results--had to be synthesized under completely anhydrous conditions. Isolation of the OA(mPEG)n conjugates was optimized by the use of ion-exchange chromatography whereby rapid removal of large amounts of uncoupled intermediate from the conjugate was achieved; the conditions of fractionation were affected by the degree of conjugation. This method of purification was superior to dialysis, ultrafiltration, and gel filtration. Furthermore, by the application of analytical hydrophobic interaction HPLC it was possible to differentiate among conjugates of different degrees of conjugation and to establish the absence of any detectable free OA in any of the preparations. The quantity of mPEG in the conjugates was determined directly by NMR.     

A sensitive multidimensional method for the detection,characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection

Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows.