Disulfide-linked Peptides Research Articles

M-nitrobenzyl alcohol supercharging reagent enhances the chromatographic separation and the charging of disulfide bond linked and His-tag peptides

The linkages of disulfide bond (DSB) play important roles in protein stability and activity. Mass spectrometry-based (MS-based) techniques become accepted tools for DSB analysis in the recent decade. In the bottom-up approach, after enzyme digestion, the neighbouring amino acids of cysteines have great impacts on the physicochemical properties of resulting disulfide bond peptides, determining their retention behaviour on liquid chromatography (LC) and their MS ionization efficiency. In this study, the addition of supercharging reagent in LC mobile phase was used to examine the impact of supercharging reagent on the charge states of disulfide-bond peptides. The results showed that 0.1 % m-nitrobenzyl alcohol (m-NBA) in LC mobile phase increased the sensitivity and charge states of DSB peptides from our model protein, equine Interleukin-5 (eIL5), as well as the resolution of reversed-phase chromatography. Notably, also the sensitivity of C-terminal peptide with His-tag significantly improved. Our findings highlight the effectiveness of employing m-NBA as a supercharging reagent when investigating disulfide-linked peptides and the C-terminal peptide with a His-tag through nano-liquid chromatography mass spectrometry.

Exploring the Potential of Bioactive Peptides: From Natural Sources to Therapeutics.

Bioactive peptides, specific protein fragments with positive health effects, are gaining traction in drug development for advantages like enhanced penetration, low toxicity, and rapid clearance. This comprehensive review navigates the intricate landscape of peptide science, covering discovery to functional characterization. Beginning with a peptidomic exploration of natural sources, the review emphasizes the search for novel peptides. Extraction approaches, including enzymatic hydrolysis, microbial fermentation, and specialized methods for disulfide-linked peptides, are extensively covered. Mass spectrometric analysis techniques for data acquisition and identification, such as liquid chromatography, capillary electrophoresis, untargeted peptide analysis, and bioinformatics, are thoroughly outlined. The exploration of peptide bioactivity incorporates various methodologies, from in vitro assays to in silico techniques, including advanced approaches like phage display and cell-based assays. The review also discusses the structure-activity relationship in the context of antimicrobial peptides (AMPs), ACE-inhibitory peptides (ACEs), and antioxidative peptides (AOPs). Concluding with key findings and future research directions, this interdisciplinary review serves as a comprehensive reference, offering a holistic understanding of peptides and their potential therapeutic applications.

The antimicrobial peptide database is 20 years old: Recent developments and future directions.

In 2023, the Antimicrobial Peptide Database (currently available at https://aps.unmc.edu) is 20-years-old. The timeline for the APD expansion in peptide entries, classification methods, search functions, post-translational modifications, binding targets, and mechanisms of action of antimicrobial peptides (AMPs) has been summarized in our previous Protein Science paper. This article highlights new database additions and findings. To facilitate antimicrobial development to combat drug-resistant pathogens, the APD has been re-annotating the data for antibacterial activity (active, inactive, and uncertain), toxicity (hemolytic and nonhemolytic AMPs), and salt tolerance (salt sensitive and insensitive). Comparison of the respective desired and undesired AMP groups produces new knowledge for peptide design. Our unification of AMPs from the six life kingdoms into "natural AMPs" enabled the first comparison with globular or transmembrane proteins. Due to the dominance of amphipathic helical and disulfide-linked peptides, cysteine, glycine, and lysine in natural AMPs are much more abundant than those in globular proteins. To include peptides predicted by machine learning, a new "predicted" group has been created. Remarkably, the averaged amino acid composition of predicted peptides is located between the lower bound of natural AMPs and the upper bound of synthetic peptides. Synthetic peptides in the current APD, with the highest cationic and hydrophobic amino acid percentages, are mostly designed with varying degrees of optimization. Hence, natural AMPs accumulated in the APD over 20 years have laid the foundation for machine learning prediction. We discuss future directions for peptide discovery. It is anticipated that the APD will continue to play a role in research and education.

Chromatography assisted in-vitro refolding and purification of recombinant peptibody: Recombinant Romiplostim a case study

In-vitro protein refolding is one of the key rate-limiting unit operations in manufacturing of fusion proteins such as peptibodies expressed using E. coli. Dilution-assisted refolding is the most commonly used industrial practice to achieve the soluble, native functional form of the recombinant protein from the inclusion bodies. This study is focused on developing a chromatography-assisted in-vitro refolding platform to produce the biologically active, native form of recombinant peptibody. Recombinant Romiplostim was selected as a model protein for the study. A plug flow tubular reactor was connected in series with capture step affinity chromatography to achieve simultaneous in-vitro refolding and capture step purification of recombinant Romiplostim. Effect of various critical process parameters like fold dilution, temperature, residence time, and Cysteine: DTT ratio was studied using a central composite based design of experiment strategy to achieve a maximum refolding yield of selected peptibody. Under optimum refolding conditions, the maximum refolding yield of 57.0 ± 1.5 % and a purity of over 79.73 ± 3.4 % were achieved at 25-fold dilution, 15 °C temperature, 6 h residence time with 6 mM and 10 mM of cysteine and DTT, respectively. The formation of native peptibody structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structure. The amino acid sequence for the disulfide-linked peptide was mapped using collision-induced dissociation (CID) to confirm the formation of interchain disulfide bonds between Cys7-Cys7 and Cys10-Cys10 similarly for intra-chain disulfide bonds between Cys42-Cys102, and Cys148-Cys206. The developed protocol here is a valuable tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter-domain disulfide bonds.

Proteomic Characterization of Native and Rearranged Disulfide Bonds in Proteins from Thermally Treated and Commercial Milk Samples.

To investigate thiol-disulfide interchange reactions in heated milk yielding non-native intramolecular rearranged and intermolecular cross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MS and dedicated bioinformatics was accomplished. Raw milk samples heated for different times and various commercial dairy products were analyzed. Qualitative experiments on tryptic digests of resolved protein mixtures assigned the corresponding disulfide-linked peptides. Results confirmed the limited data available on few milk proteins, generated the widest inventory of components (63 in number) involved in thiol-disulfide exchange processes, and provided novel structural information on S-S-bridged molecules. Quantitative experiments on unresolved protein mixtures from both sample typologies estimated the population of molecules associated with thiol-disulfide reshuffling processes. Disulfide-linked peptides associated with native intramolecular S-S bonds generally showed a progressive reduction depending on heating time/harshness, whereas those related to specific non-native intramolecular/intermolecular ones showed an opposite quantitative trend. This was associated with a temperature-dependent augmented reactivity of definite native protein thiols and S-S bridges, which determined the formation of non-native rearranged monomers and cross-linked oligomers. Results provided novel information for possibly linking the nature and extent of thiol-disulfide exchange reactions in heated milk proteins to the corresponding functional and technological characteristics, with possible implications on food digestibility, allergenicity, and bioactivity.

PDI Cleavage of Disulfide Bonds within the TP Receptor Inhibits Signaling through Gα 13

G-protein coupled receptors (GPCRs) are the most abundant superfamily of cell surface receptors. Approximately 35% of approved drugs target GPCRs and class A GPCRs account for ~85% of this superfamily. Class A GPCRs are characterized in part by two highly conserved disulfides in their extracellular domains that are thought to stabilize protein structure. Whether these disulfides can be enzymatically modified to influence G-protein signaling is not known. We and others have previously shown that disulfide bond modification by thiol isomerases such as protein disulfide isomerase (PDI) represent a previously unrecognized level of control of thrombus formation. We therefore evaluated the ability of recombinant PDI (rPDI) to modulate signaling through modification of the canonical disulfides within platelet GPCRs. Exposure of platelets to rPDI had no effect on stimulation through PAR1, PAR4, or the α 2A-adrenergic receptor. In contrast, rPDI exposure dramatically decreased platelet activation induced by the TP receptor agonists U46619 or arachidonic acid, implicating rPDI-mediated modulation of TP receptor signaling. Consistent with this finding, rPDI blocked U46619-mediated activation of α IIbβ 3, α-granule release, and dense granule release. Conversely, inhibition of endogenous PDI using either inhibitory antibodies or PDI-targeted small molecules enhanced TP receptor-mediated platelet aggregation and granule release, indicating that endogenous platelet PDI influences TP receptor signaling. Inhibition of TP receptor-mediated signaling required PDI active site cysteines since rPDI mutants lacking these cysteines lost inhibitory activity. Evaluation of the inhibitory activity of different PDI fragments showed that the PDI substrate binding domain is also critical for inhibitory activity. The ability to inhibit signaling through the TP receptor was specific for rPDI since incubation with other recombinant thiol isomerases including ERp57, ERp5, and ERp72 had no effect. To determine the specific modifications to TP receptor canonical disulfides induced by PDI, HEK cells were transfected with TP receptor and exposed to rPDI. TP receptor was subsequently immunopreciptated and disulfide-linked peptide analysis was performed using mass spectrometry. Compared to untreated controls, TP receptor exposed to rPDI demonstrated cleavage of Cys11-Cys102 and Cys105-Cys183 bonds and the generation of a new Cys102-Cys183 bond. To determine how modification of the disulfide bonding pattern affected signaling through the TP receptor, we evaluated signaling through specific Gα subunits in platelets. The platelet TP receptor signals through Gα q (which couples to phospholipase and increases calcium flux) and Gα 13 (which couples to RhoA and myosin light chain kinase). PDI-mediated cleavage of the platelet TP receptor resulted in biased signaling, with substantial inhibition of G α13-mediated RhoA-GTP activation and myosin light chain phosphorylation and little effect on Gα q-mediated calcium flux. These results show how PDI can modify platelet signaling and represent the first demonstration that a thiol isomerase can modulate the function of a GPCR via rearrangement of canonical disulfide bonds. DisclosuresNo relevant conflicts of interest to declare.

Carboxypeptidase Y Assisted Disulfide-Bond Identification with Linearized Database Search.

A disulfide bond is an important protein post-translational modification and plays a key role in regulating protein oxidation status, protein structure, and stability. Analysis of a disulfide bond using mass spectrometry is challenging because there lacks an efficient method to separate the disulfide-linked peptides from a complex protein digest, and the MS data requires sophisticated interpretation. Here, we developed a novel disulfide bond identification strategy, termed as "carboxypeptidase Y assisted disulfide-bond identification (CADI)". CADI is able to significantly reduce sample complexity by depleting ∼90% of the linear peptides while keeping the disulfide-bonded peptides. Furthermore, all CADI data can be directly analyzed by widely used protein database search engines, such as Mascot and MaxQuant. Our data show that CADI is able to sensitively identify disulfide bonds in peptides and proteins. However, CADI has not yet achieved a satisfied in-depth coverage on complex mammalian cell lysates due to the limited enzymatic activity of carboxypeptidase Y and low occurrences of disulfide bonds in a proteome. Altogether, CADI is a useful method that can get disulfide-linked peptides enriched and analyzed with regular search engines. CADI holds great potentials to deepen the analysis of disulfide bond and other types of cross-linked peptides on the proteome scale.

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.

Comprehensive Analysis of Tryptic Peptides Arising from Disulfide Linkages in NISTmAb and Their Use for Developing a Mass Spectral Library.

This work presents methods for identifying and then creating a mass spectral library for disulfide-linked peptides originating from the NISTmAb, a reference material of the humanized IgG1k monoclonal antibody (RM 8671). Analyses involved both partially reduced and non-reduced samples under neutral and weakly basic conditions followed by nanoflow liquid chromatography tandem mass spectrometry (LC–MS/MS). Spectra of peptides containing disulfide bonds are identified by both MS1 ion and MS2 fragment ion data in order to completely map all the disulfide linkages in the NISTmAb. This led to the detection of 383 distinct disulfide-linked peptide ions, arising from fully tryptic cleavage, missed cleavage, irregular cleavage, complex Met/Trp oxidation mixtures, and metal adducts. Fragmentation features of disulfide bonds under low-energy collision dissociation were examined. These include (1) peptide bond cleavage leaving disulfide bonds intact; (2) disulfide bond cleavage, often leading to extensive fragmentation; and (3) double cleavage products resulting from breakages of two peptide bonds or both peptide and disulfide bonds. Automated annotation of various complex MS/MS fragments enabled the identification of disulfide-linked peptides with high confidence. Peptides containing each of the nine native disulfide bonds were identified along with 86 additional disulfide linkages arising from disulfide bond shuffling. The presence of shuffled disulfides was nearly completely abrogated by refining digest conditions. A curated spectral library of 702 disulfide-linked peptide spectra was created from this analysis and is publicly available for free download. Since all IgG1 antibodies have the same constant regions, the resulting library can be used as a tool for facile identification of “hard-to-find” disulfide-bonded peptides. Moreover, we show that one may identify such peptides originating from IgG1 proteins in human serum, thereby serving as a means of monitoring the completeness of protein reduction in proteomics studies. Data are available via ProteomeXchange with identifier PXD023358.

Antibacterial activities of physiologically stable, self-assembled peptide nanoparticles.

In this study, we report that host defense protein-derived ten amino acid long disulfide-linked peptides self-assemble in the form of β-sheets and β-turns, and exhibit concentration-dependent self-assembly in the form of nanospheres, termed as disulfide linked nanospheres (DSNs). As expected, bare DSNs are prone to aggregation in ionic solutions and in the presence of serum proteins. To yield physiologically stable self-assembled peptide-based materials, DSNs are stabilized in the form of supramolecular assemblies using β-cyclodextrins (β-CD) and fucoidan, as delivery carriers. The inclusion complexes of DSNs with β-CD (β-CD-DSN) and electrostatic complexation of fucoidan with DSNs (FC-DSN) stabilizes the secondary structure of DSNs. Comparison of β-CD-DSNs with FC-DSNs reveals that inclusion complexes of DSNs formed in the presence of β-CD are highly stable under physiological conditions, show high cellular uptake, exhibit bacterial flocculation, and enhance antibacterial efficacies of DSNs in a range of Gram-positive and Gram-negative bacteria.

New Sea Anemone Toxin RTX-VI Selectively Modulates Voltage-Gated Sodium Channels.

A new neurotoxin RTX-VI that modulates the voltage-gated sodium channels (NaV) was isolated from the ethanolic extract of the sea anemone Heteractis crispa. Its amino acid sequence was determined using the combination of Edman degradation and tandem mass spectrometry. RTX-VI turned out to be an unusual natural analogue of the previously described sea anemone toxin RTX-III. The RTX-VI molecule consists of two disulfide-linked peptide chains and is devoid of Arg13, which is important for the selectivity and affinity of such peptides for the NaV channels. Electrophysiological screening of RTV-VI on NaV channel subtypes showed its selective interaction with the central nervous system (NaV1.2, NaV1.6) and insect (BgNaV1, VdNaV1) sodium channels.

Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using a Disulfide-Linked Peptide Reporter.

In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identified S-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond. However, the identity of the S-sulfenylated amino acid residues within a protein remained enigmatic. By using the same transgenic YAP1C probe, we present here a technological advancement to identify in situ sulfenylated cysteine sites in Arabidopsis thaliana cells under control condition and oxidative stress. Briefly, the total extract of transgenic YAP1C A. thaliana cells was initially purified on IgG-Sepharose beads, followed by a tryptic digest. Then, the mixed disulfide-linked peptides were further enriched at the peptide level on an anti-YAP1C-derived peptide (C598SEIWDR) antibody. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.

Disulfide-Linked Peptides for Blocking BTLA/HVEM Binding.

Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14–39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.

A releasable disulfide-linked peptide tag facilitates the synthesis and purification of short peptides.

Combinatorial cyclization of hundreds to thousands of random linear peptides by structurally diverse chemical linkers offers access to large macrocyclic compound libraries. A bottleneck in the development of such libraries is the preparation of large numbers of short random linear peptides. Herein, we present a tag-based strategy that is not dependent on a throughput-limiting chromatographic purification step and thus enables parallel production of short peptides. In brief, peptides are synthesized on solid phase as conjugates with a disulfide-linked Cys-Gly-Arg-Trp tetra-peptide tag. The charged arginine residue in the tag allows for purification of the peptides by diethyl ether-precipitation and the tryptophan allows for quantification of the product by absorption measurement. Addition of a reducing agent releases the short peptides from the tag. The released sulfhydryl group in the peptide can readily be used for cyclization of the peptide library with bis-electrophilic linker reagents.

A Strategy To Prepare Peptide Heterodimers in the Solid Phase with an Acid-Labile Linker.

The currently synthetic methods of peptide heterodimer involve tedious synthesis and purification steps. An acid-labile traceless linker was prepared, which is highly compatible with the Fmoc strategy and could be used to prepare peptide heterodimer on resin. The linker could be cleaved concomitantly with peptide cleavage, and two model heterodimers were synthesized. The proposed synthesis procedure is simple, straightforward, and provides great convenience for preparing disulfide-linked peptide heterodimers.

Monitoring the Disulfide Bonds of Folding Isomers of Synthetic CTX A3 Polypeptide Using MS-Based Technology.

Native disulfide formation is crucial to the process of disulfide-rich protein folding in vitro. As such, analysis of the disulfide bonds can be used to track the process of the folding reaction; however, the diverse structural isomers interfere with characterization due to the non-native disulfide linkages. Previously, a mass spectrometry (MS) based platform coupled with peptide dimethylation and an automatic disulfide bond searching engine demonstrated the potential to screen disulfide-linked peptides for the unambiguous assignment of paired cysteine residues of toxin components in cobra venom. The developed MS-based platform was evaluated to analyze the disulfide bonds of structural isomers during the folding reaction of synthetic cardiotoxin A3 polypeptide (syn-CTX A3), an important medical component in cobra venom. Through application of this work flow, a total of 13 disulfide-linked peptides were repeatedly identified across the folding reaction, and two of them were found to contain cysteine pairings, like those found in native CTX A3. Quantitative analysis of these disulfide-linked peptides showed the occurrence of a progressive disulfide rearrangement that generates a native disulfide bond pattern on syn-CTX A3 folded protein. The formation of these syn-CTX A3 folded protein reaches a steady level in the late stage of the folding reaction. Biophysical and cell-based assays showed that the collected syn-CTX A3 folded protein have a β-sheet secondary structure and cytotoxic activity similar to that of native CTX A3. In addition, the immunization of the syn-CTX A3 folded proteins could induce neutralization antibodies against the cytotoxic activity of native CTX A3. In contrast, these structure activities were poorly observed in the other folded isomers with non-native disulfide bonds. The study highlights the ability of the developed MS platform to assay isomers with heterogeneous disulfide bonds, providing insight into the folding mechanism of the bioactive protein generation.

Hinge-Type Dimerization of Proteins by a Tetracysteine Peptide of High Pairing Specificity.

Dimeric disulfide-linked peptides are formed by the regioselective oxidative folding of thiol precursors containing the CX3CX2CX3C tetracysteine motif. Here, we investigate the general applicability of this peptide as a dimerization motif for different proteins. By recombinant DNA technology, the peptide CHWECRGCRLVC was loaded with proteins, and functional homodimers were obtained upon oxidative folding. Attached to the N-terminus of the dodecapeptide, the prokaryotic enzyme limonene epoxide hydrolase (LEH) completely forms a covalent antiparallel dimer. In a diatom expression system, the monoclonal antibody CL4 mAb is released in its functional form when its natural CPPC central parallel hinge is exchanged for the designed tetra-Cys hinge motif. To improve our understanding of the regioselectivity of tetra-disulfide formation, we provoked the formation of heterodimeric hinge peptides by mixing two different tetra-Cys peptides and characterizing the heterodimer by mass spectrometry and nuclear magnetic resonance spectroscopy.

Characterization of Disulfide-Linked Peptides Using Tandem Mass Spectrometry Coupled with Automated Data Analysis Software.

Disulfide bond formation is critical for maintaining structure stability and function of many peptides and proteins. Mass spectrometry has become an important tool for the elucidation of molecular connectivity. However, the interpretation of the tandem mass spectral data of disulfide-linked peptides has been a major challenge due to the lack of appropriate tools. Developing proper data analysis software is essential to quickly characterize disulfide-linked peptides. A thorough and in-depth understanding of how disulfide-linked peptides fragment in mass spectrometer is a key in developing software to interpret the tandem mass spectra of these peptides. Two model peptides with inter- and intra-chain disulfide linkages were used to study fragmentation behavior in both collisional-activated dissociation (CAD) and electron-based dissociation (ExD) experiments. Fragments generated from CAD and ExD can be categorized into three major types, which result from different S-S and C-S bond cleavage patterns. DiSulFinder is a computer algorithm that was newly developed based on the fragmentation observed in these peptides. The software is vendor neutral and capable of quickly and accurately identifying a variety of fragments generated from disulfide-linked peptides. DiSulFinder identifies peptide backbone fragments with S-S and C-S bond cleavages and, more importantly, can also identify fragments with the S-S bond still intact to aid disulfide linkage determination. With the assistance of this software, more comprehensive disulfide connectivity characterization can be achieved. Graphical Abstract ᅟ.

Exploring ECD on a Benchtop Q Exactive Orbitrap Mass Spectrometer.

As the application of mass spectrometry intensifies in scope and diversity, the need for advanced instrumentation addressing a wide variety of analytical needs also increases. To this end, many modern, top-end mass spectrometers are designed or modified to include a wider range of fragmentation technologies, for example, ECD, ETD, EThcD, and UVPD. Still, the majority of instrument platforms are limited to more conventional methods, such as CID and HCD. While these latter methods have performed well, the less conventional fragmentation methods have been shown to lead to increased information in many applications including middle-down proteomics, top-down proteomics, glycoproteomics, and disulfide bond mapping. We describe the modification of the popular Q Exactive Orbitrap mass spectrometer to extend its fragmentation capabilities to include ECD. We show that this modification allows ≥85% matched ion intensity to originate from ECD fragment ion types as well as provides high sequence coverage (≥60%) of intact proteins and high fragment identification rates with ∼70% of ion signals matched. Finally, the ECD implementation promotes selective disulfide bond dissociation, facilitating the identification of disulfide-linked peptide conjugates. Collectively, this modification extends the capabilities of the Q Exactive Orbitrap mass spectrometer to a range of new applications.

Two snapshots of the TAPBPR-MHC I complex

Immunology Cytotoxic CD8+ T cells recognize infected and cancerous cells by scrutinizing the antigenic peptides presented by the major histocompatibility complex class I (MHC I). Peptide binding and exchange occurs in the endoplasmic reticulum in a sequence of events mediated by the chaperones tapasin and TAPBPR (see the Perspective by Cresswell). Thomas and Tampe resolved the crystal structure of the TAPBPR–MHC I editing complex by using a photocleavable high-affinity peptide to stabilize the MHC molecule. Jiang et al. crystalized MHC I molecules inhabited by truncated disulfide-linked peptides that still permit TAPBPR to bind. These complimentary snapshots elucidate the dynamic process by which chaperones stabilize the groove of peptide-free MHC I molecules. This helps MHC I sample peptide candidates and facilitates the generation of peptide repertoires enriched with high-affinity antigenic peptides. Science , this issue p. [1060][1], p. [1064][2]; see also p. [992][3] [1]: /lookup/doi/10.1126/science.aao6001 [2]: /lookup/doi/10.1126/science.aao5154 [3]: /lookup/doi/10.1126/science.aaq1398