Disulfide Mapping Research Articles

High-Coverage Disulfide Mapping Enabled by Programmable Disulfide-Ene Reaction Integrated onto a Bottom-Up Protein Analysis Workflow.

Mapping disulfide linkages is crucial for characterizing pharmaceutical proteins during drug development and quality control. Traditional bottom-up protein analysis workflows often suffer from incomplete mapping for tryptic peptides consisting of multiple disulfide bonds. Although the employment of a partial reduction of disulfide bonds can improve disulfide mapping, it becomes a bottleneck of analysis because individual tuning is often needed. Herein, we have developed an online disulfide-ene reaction system in which the composition of the reaction solvent can be programmed to achieve optimal partial reduction of tryptic disulfide peptides after liquid chromatography separation. By coupling this system onto a bottom-up protein analysis workflow, high coverage for sequencing (71-83%) and disulfide mapping (84-100%) was achieved for standard proteins consisting of 4-19 disulfide bonds. The analytical capability was further demonstrated by mapping 13 scrambled disulfide bonds in lysozyme and achieving compositional analysis of IgG isotypes (κ and λ) and subclasses (IgG1-IgG4) from human plasma.

Prolamins’ 3D structure: A new insight into protein modeling using the language of numbers and shapes

This study combined protein modeling methods to generate the prolamins’ fractions as precise as possible. Hence, gliadins, zeins, kafirins, hordeins, secalins, avenins and oryzins were generated based on their characteristics and disulfide mapping. Findings were briefly as follows: (i) Gliadins and hordeins were homologous to have both globular and rod-like structures. The ω-gliadin and C-hordein showed their worm-like structures. (ii) Zeins and kafirins had homology and existed in ordered helical-rich models. (iii) The γ-secalins exhibited similar models to that of γ-gliadin, indicating a ring connected to a tail. (iv) Avenins and Oryzins formed all possible disulfide bonds, however, there was an unusual linkage between two tandem cysteines of avenins. The studies on 3D structure of prolamins are hopefully expected to provide an improved insight at molecular level leading to accurate functionalization and applications of them in various field of studies including biology, food and medicine.

Fast and Accurate Disulfide Bridge Detection

Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multibillion dollar industry. Essential here is the quality control assessment of critical attributes, such as sequence fidelity, proper folding, and posttranslational modifications. Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition, and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis, the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background, which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within 1 h, allowing for high-throughput, high-accuracy disulfide mapping.

High-performance peptide and disulfide mapping by direct injection of intact proteins using on-line coupled UV-liquid chromatography microdroplet mass spectrometry (UVLC-MMS)

Microdroplet mass spectrometry (MMS), achieving ultra-fast enzyme digestion in the ionization source, holds great promises for innovating protein analysis. Here, in-depth protein characterization is demonstrated by direct injection of intact protein mixtures via on-line coupling MMS with capillary C4 liquid chromatography (LC) containing UV windows (UVLC-MMS) through an enzyme introduction tee. We showed complete sets of peptides of individual proteins (hemoglobin, bovine serum albumin, and ribonuclease A) in a mixture could be obtained in one injection. Such full (100%) sequence coverage, however, could not be achieved by conventional nanoLC-MS method using bottom-up approach with single enzyme. Moreover, direct injection of a chaperone α-crystalline (α-Cry) complex yielded identification of post-translational modifications including novel sites and semi-quantitative characterization including 3:1 stoichiometry ratio of αA- and αB-Cry sub-units and ∼1.4 phosphorylation/subunit on S45 (novel site) and S122 (main site) of αA-Cry, ∼0.7 phosphorylation/subunit on S19 (main site) and S45 of αB-Cry, as well as 100% acetylation on both N-termini of each subunits by matching the mass and retention time of the intact and its digested peptides. Furthermore, trifluoroacetic acid was able to be used in the mobile phase with UVLC-MMS to improve the separation of differentially reduced intact species and detectability of the droplet-digested products. This allowed us to completely map four disulfide linkages of ribonuclease A based on collision-induced dissociation of disulfide clusters, some of which would otherwise not be detected, preventing scrambling or shuffling errors arising from lengthy bulk solution digestion by the bottom-up approach. Integration of UVLC and MMS greatly improves droplet digestion efficiency and MS detection, enabling highly efficient workflow for in-depth and accurate protein characterization.

Ginsentide-like Coffeetides Isolated from Coffee Waste Are Cell-Penetrating and Metal-Binding Microproteins.

Coffee processing generates a huge amount of waste that contains many natural products. Here, we report the discovery of a panel of novel cell-penetrating and metal ion-binding microproteins designated coffeetide cC1a-c and cL1-6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and a database search, we show that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif, but without a chitin-binding domain. NMR determination of cC1a reveals a compact structure that confers its resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis reveals that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement, like ginsentide precursors. Molecular modeling, together with experimental validation, revealed a Mg2+ and Fe3+ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have the potential to target intracellular proteins.

Understanding unfolding and refolding of the antibody fragment (Fab) III: Mapping covalent and non-covalent interactions during in-vitro refolding of light chain, heavy chain, and Fab.

In-vitro refolding of the antibody fragments from inclusion bodies is a critical manufacturing bottleneck. We have previously reported that Fab refolding involves the formation of an intermediate. To further decode the refolding pathway, we have mapped the covalent and non-covalent interactions during in-vitro refolding of individual light chain (LC), heavy chain (HC), and intact Fab. Intrinsic fluorescence analysis was performed to map the non-covalent interactions, whereas time-dependent disulfide mapping was performed using LC-MS/MS analysis under non-reducing and reducing atmospheres. Refolding of purified LC, HC, and intact Fab (Biosimilar rHu Ranibizumab) was investigated in the study. Under the reducing condition, LC, HC, and intact Fab required around 96, 30, and 70 h, respectively, whereas, under non-reducing conditions, they required only 48, 10, and 48 h, respectively, to acquire the native conformation. The disulfide bond mapping revealed that the bond between LC_Cys23/Cys88 is more unstable. The peptides mapped with the inter-chain disulfide bond were identified at 48 h during the refolding and can be considered as the rate-limiting step in (Fab) refolding. We have also studied a new refolding strategy involving assembly of separately refolded LC and HC into Fab molecule. In this case the Fab refolding yield was low (18.0 ± 1.36%), indicating inefficient assembly of refolded LC and HC chain into native Fab molecule.

Fuzzy supertertiary interactions within PSD-95 enable ligand binding.

The scaffold protein PSD-95 links postsynaptic receptors to sites of presynaptic neurotransmitter release. Flexible linkers between folded domains in PSD-95 enable a dynamic supertertiary structure. Interdomain interactions within the PSG supramodule, formed by PDZ3, SH3, and Guanylate Kinase domains, regulate PSD-95 activity. Here we combined discrete molecular dynamics and single molecule Förster resonance energy transfer (FRET) to characterize the PSG supramodule, with time resolution spanning picoseconds to seconds. We used a FRET network to measure distances in full-length PSD-95 and model the conformational ensemble. We found that PDZ3 samples two conformational basins, which we confirmed with disulfide mapping. To understand effects on activity, we measured binding of the synaptic adhesion protein neuroligin. We found that PSD-95 bound neuroligin well at physiological pH while truncated PDZ3 bound poorly. Our hybrid structural models reveal how the supertertiary context of PDZ3 enables recognition of this critical synaptic ligand.

Mass Spectrometry-Based Disulfide Mapping of Lysyl Oxidase-like 2.

Lysyl oxidase-like 2 (LOXL2) catalyzes the oxidative deamination of peptidyl lysines and hydroxylysines to promote extracellular matrix remodeling. Aberrant activity of LOXL2 has been associated with organ fibrosis and tumor metastasis. The lysine tyrosylquinone (LTQ) cofactor is derived from Lys653 and Tyr689 in the amine oxidase domain via post-translational modification. Based on the similarity in hydrodynamic radius and radius of gyration, we recently proposed that the overall structures of the mature LOXL2 (containing LTQ) and the precursor LOXL2 (no LTQ) are very similar. In this study, we conducted a mass spectrometry-based disulfide mapping analysis of recombinant LOXL2 in three forms: a full-length LOXL2 (fl-LOXL2) containing a nearly stoichiometric amount of LTQ, Δ1-2SRCR-LOXL2 (SRCR1 and SRCR2 are truncated) in the precursor form, and Δ1-3SRCR-LOXL2 (SRCR1, SRCR2, SRCR3 are truncated) in a mixture of the precursor and the mature forms. We detected a set of five disulfide bonds that is conserved in both the precursor and the mature recombinant LOXL2s. In addition, we detected a set of four alternative disulfide bonds in low abundance that is not associated with the mature LOXL2. These results suggest that the major set of five disulfide bonds is retained post-LTQ formation.

Photochemical Disulfide-Ene Modification Enhances Protein Sequencing and Disulfide Mapping by Mass Spectrometry.

A new photochemical disulfide-ene reaction system capable of alkylating protein disulfide bonds in seconds has been established. The system is simple, containing acetone and isopropanol for disulfide reduction under 254 nm UV irradiation and norbornene as a highly efficient alkylation reagent. Enhanced characterization of disulfide-rich proteins with significantly shortened analysis time is demonstrated by coupling the reaction online with mass spectrometry.

Rapid Disulfide Mapping in Peptides and Proteins by meta-Chloroperoxybenzoic Acid (mCPBA) Oxidation and Tandem Mass Spectrometry.

Disulfide bonds are a class of important post-translational modifications that play important roles in modulating the structures and functions of proteins. Therefore, the mapping of disulfide linkages in peptides and proteins is indispensable for complete structure characterization and functional studies. As disulfide bonds in protonated ions do not dissociate readily under low-energy collision-induced dissociation (CID), they are usually chemically cleaved or activated prior to mass spectrometry (MS) or tandem MS (MS/MS) analysis. In this study, we report a new method that allows the mapping of disulfide linkages in peptides and proteins through meta-chloroperoxybenzoic acid (mCPBA)-based disulfide oxidation and MS/MS. Upon oxidation, the disulfide bond is converted to a thiosulfinate group, i.e., S(═O)-S, in a rapid (>60% yield in 1 min) and highly specific approach in an aqueous phase. The thiosulfinate group is then preferentially cleaved by MS/MS. For interchain disulfide linkages, this leads to a facile peptide chain separation and the identification of disulfide-linked peptides. For intrachain disulfide linkages, collisional activation of the thiosulfinate leads to disulfide cleavage and fragmentation of the peptide backbone constrained by the disulfide loop, enabling a near-complete peptide sequencing. The mCPBA oxidation-based disulfide mapping strategy can be readily integrated with bottom-up or top-down protein analysis for comprehensive protein structure elucidation, e.g., digested lysozyme and intact human insulin.

Raman Metrics for Molybdenum Disulfide and Graphene Enable Statistical Mapping of Nanosheet Populations

The growing research interest and uptake of layered nanomaterials for real-world applications require efficient, reliable, high-quality characterization methods. Liquid-exfoliated graphene has well...

Potentides: New Cysteine-Rich Peptides with Unusual Disulfide Connectivity from Potentilla anserina.

Cysteine-rich peptides (CRPs), which are disulfide-constrained peptides with 3 to 5 disulfide bonds and molecular weights of 2 to 6 kDa, are generally hyperstable and resistant to thermal, chemical, and enzymatic degradation. Herein, the discovery and characterization of a novel suite of CRPs, collectively named potentides pA1-pA16 from the root of the medicinal herb Potentilla anserina L, are described. Through a combination of proteomic and transcriptomic methods, it is shown that 35-residue potentide pA3, which is the most abundant member of potentides, exhibits high stability against heat, acidic, and proteolytic degradation. Transcriptomic analysis revealed that potentide precursor sequences contained four tandem repeats in the mature domain, which is the first report on tandem repeats being found in the Rosaceae family. Disulfide mapping showed that potentide pA3 displayed a novel disulfide connectivity of C1-C3, C2-C6, and C4-C5; a cystine motif that has not been reported in plant CRPs. Transcriptomic data mining and a neighbor-joining clustering analysis revealed 56 potentide homologues and their distribution in the families of Rosaceae and Ranunculaceae in angiosperm. Altogether, these results reveal a new plant CRP structure with an unusual cystine connectivity. Additionally, this study expands the families and structure diversity of CRPs as potentially active peptide pharmaceuticals.

Ginsentides: Cysteine and Glycine-rich Peptides from the Ginseng Family with Unusual Disulfide Connectivity

Ginseng, a popular and valuable traditional medicine, has been used for centuries to maintain health and treat disease. Here we report the discovery and characterization of ginsentides, a novel family of cysteine and glycine-rich peptides derived from the three most widely-used ginseng species: Panax ginseng, Panax quinquefolius, and Panax notoginseng. Using proteomic and transcriptomic methods, we identified 14 ginsentides, TP1-TP14 which consist of 31–33 amino acids and whose expression profiles are species- and tissues-dependent. Ginsentides have an eight-cysteine motif typical of the eight-cysteine-hevein-like peptides (8C-HLP) commonly found in medicinal herbs, but lack a chitin-binding domain. Transcriptomic analysis showed that the three-domain biosynthetic precursors of ginsentides differ from known 8C-HLP precursors in architecture and the absence of a C-terminal protein-cargo domain. A database search revealed an additional 50 ginsentide-like precursors from both gymnosperms and angiosperms. Disulfide mapping and structure determination of the ginsentide TP1 revealed a novel disulfide connectivity that differs from the 8C-HLPs. The structure of ginsentide TP1 is highly compact, with the N- and C-termini topologically fixed by disulfide bonds to form a pseudocyclic structure that confers resistance to heat, proteolysis, and acid and serum-mediated degradation. Together, our results expand the chemical space of natural products found in ginseng and highlight the occurrence, distribution, disulfide connectivity, and precursor architectures of cysteine- and glycine-rich ginsentides as a class of novel non-chitin-binding, non-cargo-carrying 8C-HLPs.

Acetone/Isopropanol Photoinitiating System Enables Tunable Disulfide Reduction and Disulfide Mapping via Tandem Mass Spectrometry.

Herein, we report the development of a new photochemical system which enables rapid and tunable disulfide bond reduction and its application in disulfide mapping via online coupling with mass spectrometry (MS). Acetone, a clean and electrospray ionization (ESI) compatible solvent, is used as the photoinitiator (1% volume) in the solvent system consisting of 1:1 alkyl alcohol and water. Under ultraviolet (UV) irradiation (∼254 nm), the acetone/alcohol system produces hydroxyalkyl radicals, which are responsible for disulfide bond cleavage in peptides. Acetone/isopropanol is most suitable for optimizing the disulfide reduction products, leading to almost complete conversion in less than 5 s when the reaction is conducted in a flow microreactor. The flow microreactor device not only facilitates direct coupling with ESI-MS but also allows fine-tuning of the extent of disulfide reduction by varying the UV exposure time. Near full sequence coverage for peptides consisting of intra- or interchain disulfide bonds has been achieved from complete disulfide reduction and online tandem mass spectrometry (MS/MS) via low energy collision-induced dissociation. Coupling different degrees of partial disulfide reduction with ESI-MS/MS allows disulfide mapping as demonstrated for characterizing the three disulfide bonds in insulin.

Generic Workflow for Mapping of Complex Disulfide Bonds Using In-Source Reduction and Extracted Ion Chromatograms from Data-Dependent Mass Spectrometry.

Disulfide bond mapping is a critical task in protein characterization as protein stability, structure, and function is dependent on correct cysteine connectivities. Mass spectrometry (MS) is the method of choice for this, providing fast and accurate characterization of simple disulfide bonds. Disulfide mapping by liquid chromatography tandem mass spectrometry (LC-MS/MS) is performed by identifying disulfide-bonded partner peptides following proteolytic digestion. With the recently introduced ability to assign complex disulfide patterns by online postcolumn partial disulfide reduction by in-source reduction (ISR) in a LC-ISR-MS/MS methodology, the main challenge is data analysis to ensure detection of both expected and unexpected disulfide species. In this study, we introduced a workflow for confident and unbiased mapping of complex disulfide bonds using the powerful combination of extracted ion chromatograms (XICs) of LC-ISR-MS/MS data. With postcolumn partial reduction, identical LC retention times of intact disulfide-bonded species, their constituting free peptides, and partially reduced variants were observed. Subsequent selective MS/MS fragmentation of all reduction products allowed confident identification of free cysteine-containing peptides using a classical shotgun proteomics database search. Matching XICs of the identified cysteine-containing peptides allowed identification of both predicted and unpredicted disulfide species, including unforeseen proteolytic specificities, missed cleavage sites, scrambled disulfide variants, and the presence of disulfide-entangled complexes. Applying this workflow, we successfully mapped the complex disulfide bonds of tertiapin and the epidermal growth factor (EGF) family members transforming growth factor α (TGFα) and EGF. In addition, we were able to characterize the disulfide patterns of the special disulfide fold of the TGFβ superfamily in an all-online methodology.

Mapping disulfide bonds from sub-micrograms of purified proteins or micrograms of complex protein mixtures

Disulfide bonds are vital for protein functions, but locating the linkage sites has been a challenge in protein chemistry, especially when the quantity of a sample is small or the complexity is high. In 2015, our laboratory developed a sensitive and efficient method for mapping protein disulfide bonds from simple or complex samples (Lu et al. in Nat Methods 12:329, 2015). This method is based on liquid chromatography–mass spectrometry (LC–MS) and a powerful data analysis software tool named pLink. To facilitate application of this method, we present step-by-step disulfide mapping protocols for three types of samples—purified proteins in solution, proteins in SDS-PAGE gels, and complex protein mixtures in solution. The minimum amount of protein required for this method can be as low as several hundred nanograms for purified proteins, or tens of micrograms for a mixture of hundreds of proteins. The entire workflow—from sample preparation to LC–MS and data analysis—is described in great detail. We believe that this protocol can be easily implemented in any laboratory with access to a fast-scanning, high-resolution, and accurate-mass LC–MS system.

Cystamine and Disulfiram Inhibit Human Transglutaminase 2 via an Oxidative Mechanism.

The catalytic activity of transglutaminase 2 (TG2), a ubiquitously expressed mammalian enzyme, is regulated by multiple post-translational mechanisms. Because elevated activity of TG2 in the extracellular matrix is associated with organ-specific diseases such as celiac disease and renal fibrosis, there is growing therapeutic interest in inhibitors of this enzyme. Cystamine, a symmetric disulfide compound, is one of the earliest reported TG2 inhibitors. Despite its widespread use as a tool compound to block TG2 activity in vitro and in vivo, its mechanism of action has remained unclear. Here, we demonstrate that cystamine irreversibly inhibits human TG2 ( kinh/ Ki = 1.2 mM-1 min-1) via a mechanism fundamentally distinct from those proposed previously. Through mass spectrometric disulfide mapping and site-directed mutagenesis, we show that cystamine promotes the formation of a physiologically relevant disulfide bond between Cys370 and Cys371 that allosterically abrogates the catalytic activity of human TG2. This discovery led us to evaluate clinically useful thiol → disulfide oxidants for TG2 inhibitory activity. It is demonstrated that disulfiram, a relatively safe oral thiuram disulfide, is a fairly potent TG2 inhibitor ( kinh/ Ki = 8.3 mM-1 min-1) and may therefore provide a practical tool for clinically validating this emerging therapeutic target in intestinal disorders such as celiac disease.

We FRET so You Don't Have To: New Models of the Lipoprotein Lipase Dimer.

Lipoprotein lipase (LPL) is a dimeric enzyme that is responsible for clearing triglyceride-rich lipoproteins from the blood. Although LPL plays a key role in cardiovascular health, an experimentally derived three-dimensional structure has not been determined. Such a structure would aid in understanding mutations in LPL that cause familial LPL deficiency in patients and help in the development of therapeutic strategies to target LPL. A major obstacle to structural studies of LPL is that LPL is an unstable protein that is difficult to produce in the quantities needed for nuclear magnetic resonance or crystallography. We present updated LPL structural models generated by combining disulfide mapping, computational modeling, and data derived from single-molecule Förster resonance energy transfer (smFRET). We pioneer the technique of smFRET for use with LPL by developing conditions for imaging active LPL and identifying positions in LPL for the attachment of fluorophores. Using this approach, we measure LPL-LPL intermolecular interactions to generate experimental constraints that inform new computational models of the LPL dimer structure. These models suggest that LPL may dimerize using an interface that is different from the dimerization interface suggested by crystal packing contacts seen in structures of pancreatic lipase.

Simplified identification of disulfide, trisulfide, and thioether pairs with 213 nm UVPD.

Disulfide heterogeneity and other non-native crosslinks introduced during therapeutic antibody production and storage could have considerable negative effects on clinical efficacy, but tracking these modifications remains challenging. Analysis must also be carried out cautiously to avoid introduction of disulfide scrambling or reduction, necessitating the use of low pH digestion with less specific proteases. Herein we demonstrate that 213 nm ultraviolet photodissociation streamlines disulfide elucidation through bond-selective dissociation of sulfur-sulfur and carbon-sulfur bonds in combination with less specific backbone dissociation. Importantly, both types of fragmentation can be initiated in a single MS/MS activation stage. In addition to disulfide mapping, it is also shown that thioethers and trisulfides can be identified by characteristic fragmentation patterns. The photochemistry resulting from 213 nm excitation facilitates a simplified, two-tiered data processing approach that allows observation of all native disulfide bonds, scrambled disulfide bonds, and non-native sulfur-based linkages in a pepsin digest of Rituximab. Native disulfides represented the majority of bonds according to ion count, but the highly solvent-exposed heavy/light interchain disulfides were found to be most prone to modification. Production and storage methods that facilitate non-native links are discussed. Due to the importance of heavy and light chain connectivity for antibody structure and function, this region likely requires particular attention in terms of its influence on maintaining structural fidelity.

DIsulfide Mapping PLanner Software Tool

Disulfide bridges are side-chain-mediated covalent bonds between cysteines that stabilize many protein structures. Disulfide mapping experiments to resolve these linkages typically involve proteolytic cleavage of the protein of interest followed by mass spectroscopy to identify fragments corresponding to linked peptides. Here we report the sequence-based "DIMPL" web tool to facilitate the planning and analysis steps of experimental mapping studies. The software tests permutations of user-selected proteases to determine an optimal peptic digest that produces cleavage between cysteine residues, thus separating each to an individual peptide fragment. The webserver returns fragment sequence and mass data that can be dynamically ordered to enable straightforward comparative analysis with mass spectroscopy results, facilitating dipeptide identification.