Mapping Isomeric Peptides Derived from Biopharmaceuticals Using High-Resolution Ion Mobility Mass Spectrometry.

The identification and localization of isomeric peptide modifications is a critical requirement of the biopharmaceutical industry. Despite the ability of liquid chromatography-mass spectrometry to identify many of the common post translational modifications, the identification of isobaric or racemized peptides is confounded by modern mass spectrometry-based techniques. Here, we present a novel approach combining liquid chromatography with a high-resolution ion mobility mass spectrometry system to differentiate peptide and peptide fragments based upon their mobility and mass.

Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin.

Human α2-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M's unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible "bait region." As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the "tabula rasa" bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.

Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism.

Human α2-macroglobulin (A2M) is the most characterized protease inhibitor in the alpha-macroglobulin (αM) superfamily, but the structure of its native conformation has not been determined. Here, we combined negative stain electron microscopy (EM), small-angle X-ray scattering (SAXS), and cross-linking-mass spectrometry (XL-MS) to investigate native A2M and its collapsed conformations that are obtained through aminolysis of its thiol ester by methylamine or cleavage of its bait region by trypsin. The combined interpretation of these data resulted in a model of the native A2M tetramer and its conformational changes. Native A2M consists of two crescent-shaped disulfide-bridged subunit dimers, which face toward each other and surround a central hollow space. In native A2M, interactions across the disulfide-bridged dimers are minimal, with a single major interface between the linker (LNK) regions of oppositely positioned subunits. Bait region cleavage induces both intrasubunit domain repositioning and an altered configuration of the disulfide-bridged dimer. These changes collapse the tetramer into a more compact conformation, which encloses an interior protease-trapping cavity. A recombinant A2M with a modified bait region was used to map the bait region's position in native A2M by XL-MS. A second recombinant A2M introduced an intersubunit disulfide into the LNK region, demonstrating the predicted interactions between these regions in native A2M. Altogether, our native A2M model provides a structural foundation for understanding A2M's protease-trapping mechanism, its conformation-dependent receptor interactions, and the dissociation of native A2M into dimers due to inflammatory oxidative stress.

Engineering of Orally Available, Ultralong-Acting Insulin Analogues: Discovery of OI338 and OI320.

Recently, the first basal oral insulin (OI338) was shown to provide similar treatment outcomes to insulin glargine in a phase 2a clinical trial. Here, we report the engineering of a novel class of basal oral insulin analogues of which OI338, 10, in this publication, was successfully tested in the phase 2a clinical trial. We found that the introduction of two insulin substitutions, A14E and B25H, was needed to provide increased stability toward proteolysis. Ultralong pharmacokinetic profiles were obtained by attaching an albumin-binding side chain derived from octadecanedioic (C18) or icosanedioic acid (C20) to the lysine in position B29. Crucial for obtaining the ultralong PK profile was also a significant reduction of insulin receptor affinity. Oral bioavailability in dogs indicated that C18-based analogues were superior to C20-based analogues. These studies led to the identification of the two clinical candidates OI338 and OI320 (10 and 24, respectively).

Characterization of Insulin Dimers by Top-Down Mass Spectrometry.

High-molecular weight products (HMWP) are an important critical quality attribute in research and development of insulin biopharmaceuticals. We here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites. On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains. The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links. Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds. UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer. Thus, using varied multistage approaches, it was discovered that in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain. In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-. The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds. Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Substituting the Thiol Ester of Human A2M or C3 with a Disulfide Produces Native Proteins with Altered Proteolysis-Induced Conformational Changes.

Most proteins in the α-macroglobulin (αM) superfamily contain reactive thiol esters that are required for their biological function. Here, we have characterized the human α2-macroglobulin (A2M) and complement component C3 mutants A2M Q975C and C3 Q1013C, which replace the CGEQ thiol ester motifs of the original proteins with the disulfide-forming sequence CGEC. Mass spectrometry showed that the intended disulfide was formed in both proteins. The correct folding and native conformation of A2M Q975C were shown by its assembly to a tetramer, an initially slow electrophoretic mobility with a demonstrable conformational collapse induced by proteolysis, functional protease trapping, and conformation-dependent interactions with low-density lipoprotein receptor-related protein 1. However, A2M Q975C had a decreased capacity to inhibit trypsin and was more susceptible to cleavage by trypsin or thermolysin when compared to wild-type A2M. C3 Q1013C also folded correctly and was initially in a native conformation, as demonstrated by its cation exchange elution profile, electrophoretic mobility, and interaction with complement factor B, although it assumed a conformation that was distinct from native C3, C3b, or C3(H2O) when cleaved by trypsin. These results demonstrate that disulfides can substitute thiol esters and maintain the native conformations of A2M and C3. Additionally, they indicate that proteolysis is not the sole factor in the conformational changes of A2M and C3 and that thiol ester lysis also plays a role.

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

Molecular engineering of safe and efficacious oral basal insulin.

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.

Direct Ultraviolet Laser-Induced Reduction of Disulfide Bonds in Insulin and Vasopressin.

Ultraviolet (UV) light has been shown to induce reduction of disulfide bonds in proteins in solution. The photoreduction is proposed to be a result of electron donation from excited Tyr or Trp residues. In this work, a powerful UV femtosecond laser was used to generate photoreduced products, while the hypothesis of Tyr/Trp mediation was studied with spectroscopy and mass spectrometry. With limited irradiation times of 3 min or less at 280 nm, the laser-induced reduction in arginine vasopressin and human insulin led to significant yields of ∼3% stable reduced product. The photogenerated thiols required acidic pH for stabilization, while neutral pH primarily caused scrambling and trisulfide formation. Interestingly, there was no direct evidence that Tyr/Trp mediation was a required criterion for the photoreduction of disulfide bonds. Intermolecular electron transfer remained a possibility for insulin but was ruled out for vasopressin. We propose that an additional mechanism should be increasingly considered in UV light-induced reduction of disulfide bonds in solution, in which a single UV photon is directly absorbed by the disulfide bond.

Characterization of Ultraviolet Photoreactions in Therapeutic Peptides by Femtosecond Laser Catalysis and Mass Spectrometry.

Peptides and proteins have diverse ultraviolet (UV) photoreaction pathways that can be activated by the energy of the UV photons absorbed. Simple light sources such as lamps are conventionally used to study these photoreactions in solution. This work provides a proof of concept that femtosecond laser technology can function as a highly potent UV source in rapidly conducting UV photostability studies of peptides. Correspondingly, sufficient quantities of photoproducts were generated in 1 min or less, allowing for identification of known and new photomodifications in the therapeutic peptides somatostatin-14 and arginine vasopressin. Identical photoproducts were also generated with a conventional continuous source. The major modifications included N-formylkynurenine, a cross-link between Trp and Phe, a Tyr product with an NH3 loss, and disruption of an unstable disulfide bond into a complex mixture of a trisulfide bond and multiple scrambled dimeric products. In conclusion, femtosecond lasers are extremely useful to drive fast UV-induced reactions for high throughput screening of photostability and modifications in amino acid polymers.

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 TGFß superfamily in an all-online methodology.

Complete Mapping of Complex Disulfide Patterns with Closely-Spaced Cysteines by In-Source Reduction and Data-Dependent Mass Spectrometry.

Mapping of disulfide bonds is an essential part of protein characterization to ensure correct cysteine pairings. For this, mass spectrometry (MS) is the most widely used technique due to fast and accurate characterization. However, MS-based disulfide mapping is challenged when multiple disulfide bonds are present in complicated patterns. This includes the presence of disulfide bonds in nested patterns and closely spaced cysteines. Unambiguous mapping of such disulfide bonds typically requires advanced MS approaches. In this study, we exploited in-source reduction (ISR) of disulfide bonds during the electrospray ionization process to facilitate disulfide bond assignments. We successfully developed a LC-ISR-MS/MS methodology to use as an online and fully automated partial reduction procedure. Postcolumn partial reduction by ISR provided fast and easy identification of peptides involved in disulfide bonding from nonreduced proteolytic digests, due to the concurrent detection of disulfide-containing peptide species and their composing free peptides. Most importantly, intermediate partially reduced species containing only a single disulfide bond were also generated, from which unambiguous assignment of individual disulfide bonds could be done in species containing closely spaced disulfide bonds. The strength of this methodology was demonstrated by complete mapping of all four disulfide bonds in lysozyme and all 17 disulfide bonds in human serum albumin, including nested disulfide bonds and motifs of adjacent cysteine residues.

Electron Transfer Dissociation of All Ions at All Times, MSETD, in a Quadrupole Time-of-Flight (Q-ToF) Mass Spectrometer.

Data-independent mass spectral acquisition is particularly powerful when combined with ultra-performance liquid chromatography (LC) that provides excellent separation of most components present in a given sample. Data-independent analysis (DIA) consists of alternating full MS scans and scans with fragmentation of all ions within a selected m/z range, providing precursor masses and structure information, respectively. Fragmentation spectra are acquired either by sequential isolation and fragmentation of sliding m/z ranges or fragmenting all ions entering the MS instrument with no ion isolation, termed broadband DIA. Previously, broadband DIA has only been possible using collision induced dissociation (CID). Here, we report the use of electron transfer dissociation (ETD) as the fragmentation technique in broadband DIA instead of traditional collision induced dissociation (CID) during MSE. In this approach, which we refer to as MSETD, we implement the inherent benefits provided by ETD, such as discrimination of leucine and isoleucine, in a DIA setup. The combination of DIA analysis and ETD fragmentation with supplemental CID energy provides a powerful platform to obtain information on all precursors and their sequence from a single experiment. Graphical Abstract ᅟ.

Disulfide Linkage Characterization of Disulfide Bond-Containing Proteins and Peptides by Reducing Electrochemistry and Mass Spectrometry.

Unravelling of disulfide linkage patterns is a crucial part of protein characterization, whether it is for a previously uncharacterized protein in basic research or a recombinant pharmaceutical protein. In the biopharmaceutical industry, elucidation of the cysteine connectivities is a necessity to avoid disulfide scrambled and incorrectly folded forms in the final product. Mass spectrometry (MS) is a highly utilized analytical tool for this due to fast and accurate characterization. However, disulfide bonds being an additional covalent bond in the protein structure represent a challenge in protein sequencing by tandem MS (MS/MS). Electrochemical (EC) reduction of disulfide bonds has recently been demonstrated to provide efficient reduction efficiencies, significantly enhancing sequence coverages in online coupling with MS characterization. In this study, the potential use of EC disulfide reduction in combination with MS characterization for disulfide mapping was assessed. We employed two approaches based on (1) the high flexibility and instant information about the degree of reduction in infusion EC-MS to generate partially reduced species on the intact protein level and (2) the preserved link between parent disulfide-linked fragments and free reduced peptides in an LC-EC-MS platform of nonreduced proteolytic protein digestions. Here we report the successful use of EC as a partial reduction approach in mapping of disulfide bonds of intact human insulin (HI) and lysozyme. In addition, we established a LC-EC-MS platform advantageous in disulfide characterization of complex and highly disulfide-bonded proteins such as human serum albumin (HSA) by online EC reduction of nonreduced proteolytic digestions.