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Through mechanistic work and rational design, we have developed the fastest organometallic abiotic Cys bioconjugation. As a result, the developed organometallic Au(III) bioconjugation reagents enable selective labeling of Cys moieties down to picomolar concentrations and allow for the rapid construction of complex heterostructures from peptides, proteins, and oligonucleotides. This work showcases how organometallic chemistry can be interfaced with biomolecules and lead to a range of reactivities that are largely unmatched by classical organic chemistry tools.

Amyloid fibrils of tau are increasingly accepted as a cause of neuronal death and brain atrophy in Alzheimer's disease (AD). Diminishing tau aggregation is a promising strategy in the search for efficacious AD therapeutics. Previously, our laboratory designed a six-residue, nonnatural amino acid inhibitor D-TLKIVW peptide (6-DP), which can prevent tau aggregation in vitro. However, it cannot block cell-to-cell transmission of tau aggregation. Here, we find D-TLKIVWC (7-DP), a d-cysteine extension of 6-DP, not only prevents tau aggregation but also fragments tau fibrils extracted from AD brains to neutralize their seeding ability and protect neuronal cells from tau-induced toxicity. To facilitate the transport of 7-DP across the blood-brain barrier, we conjugated it to magnetic nanoparticles (MNPs). The MNPs-DP complex retains the inhibition and fragmentation properties of 7-DP alone. Ten weeks of MNPs-DP treatment appear to reverse neurological deficits in the PS19 mouse model of AD. This work offers a direction for development of therapies to target tau fibrils.

Despite much effort, antibody therapies for Alzheimers disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are two of the most important therapeutic drug classes that require extensive characterization, whereas their large size and structural complexity make them challenging to characterize and demand the use of advanced analytical methods. Top-down mass spectrometry (TD-MS) is an emerging technique that minimizes sample preparation and preserves endogenous post-translational modifications (PTMs); however, TD-MS of large proteins suffers from low fragmentation efficiency, limiting the sequence and structure information that can be obtained. Here, we show that including the assignment of internal fragments in native TD-MS of an intact mAb and an ADC can improve their molecular characterization. For the NIST mAb, internal fragments can access the sequence region constrained by disulfide bonds to increase the TD-MS sequence coverage to over 75%. Important PTM information, including intrachain disulfide connectivity and N-glycosylation sites, can be revealed after including internal fragments. For a heterogeneous lysine-linked ADC, we show that assigning internal fragments improves the identification of drug conjugation sites to achieve a coverage of 58% of all putative conjugation sites. This proof-of-principle study demonstrates the potential value of including internal fragments in native TD-MS of intact mAbs and ADCs, and this analytical strategy can be extended to bottom-up and middle-down MS approaches to achieve even more comprehensive characterization of important therapeutic molecules.

Synucleinopathies are a group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). These diseases are characterized by the aggregation and deposition of α-synuclein (α-syn) in Lewy bodies (LBs) in PD and DLB or as glial cytoplasmic inclusions in MSA. In healthy brains, only ∼4% of α-syn is phosphorylated at Ser129 (pS129-α-syn), whereas >90% pS129-α-syn may be found in LBs, suggesting that pS129-α-syn could be a useful biomarker for synucleinopathies. However, a widely available, robust, sensitive, and reproducible method for measuring pS129-α-syn in biological fluids is currently missing. We used Meso Scale Discovery (MSD)'s electrochemiluminescence platform to create a new assay for sensitive detection of pS129-α-syn. We evaluated several combinations of capture and detection antibodies and used semisynthetic pS129-α-syn as a standard for the assay at a concentration range from 0.5 to 6.6 × 104 pg/mL. Using the antibody EP1536Y for capture and an anti-human α-syn antibody (MSD) for detection was the best combination in terms of assay sensitivity, specificity, and reproducibility. We tested the utility of the assay for the detection and quantification of pS129-α-syn in human cerebrospinal fluid, serum, plasma, saliva, and CNS-originating small extracellular vesicles, as well as in mouse brain lysates. Our data suggest that the assay can become a widely used method for detecting pS129-α-syn in biomedical studies including when only a limited volume of sample is available and high sensitivity is required, offering new opportunities for diagnostic biomarkers, monitoring disease progression, and quantifying outcome measures in clinical trials.

Streptococcus pyogenes (group A Streptococcus) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections, S. pyogenes uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains. Based on the results of X-ray crystallography, small angle X-ray scattering, NMR spectroscopy, native mass spectrometry, and heme transfer experiments, we propose that Shr utilizes a "cap and release" mechanism to gather heme from Hb. In the mechanism, Shr uses the HID1 and HID2 modules to preferentially recognize only heme-loaded forms of Hb by contacting the edges of its protoporphyrin rings. Heme transfer is enabled by significant receptor dynamics within the Shr-Hb complex which function to transiently uncap HID1 from the heme bound to Hb's β subunit, enabling the gated release of its relatively weakly bound heme molecule and subsequent capture by Shr's NEAT domains. These dynamics may maximize the efficiency of heme scavenging by S. pyogenes, enabling it to preferentially recognize and remove heme from only heme-loaded forms of Hb that contain iron.

Disulfide bonds in proteins have a substantial impact on protein structure, stability, and biological activity. Localizing disulfide bonds is critical for understanding protein folding and higher-order structure. Conventional top-down mass spectrometry (TD-MS), where only terminal fragments are assigned for disulfide-intact proteins, can access disulfide information, but suffers from low fragmentation efficiency, thereby limiting sequence coverage. Here, we show that assigning internal fragments generated from TD-MS enhances the sequence coverage of disulfide-intact proteins by 20-60% by returning information from the interior of the protein sequence, which cannot be obtained by terminal fragments alone. The inclusion of internal fragments can extend the sequence information of disulfide-intact proteins to near complete sequence coverage. Importantly, the enhanced sequence information that arise from the assignment of internal fragments can be used to determine the relative position of disulfide bonds and the exact disulfide connectivity between cysteines. The data presented here demonstrates the benefits of incorporating internal fragment analysis into the TD-MS workflow for analyzing disulfide-intact proteins, which would be valuable for characterizing biotherapeutic proteins such as monoclonal antibodies and antibody-drug conjugates.

Native mass spectrometry (MS) of proteins and protein assemblies reveals size and binding stoichiometry, but elucidating structures to understand their function is more challenging. Native top-down MS (nTDMS), i.e., fragmentation of the gas-phase protein, is conventionally used to derive sequence information, locate post-translational modifications (PTMs), and pinpoint ligand binding sites. nTDMS also endeavors to dissociate covalent bonds in a conformation-sensitive manner, such that information about higher-order structure can be inferred from the fragmentation pattern. However, the activation/dissociation method used can greatly affect the resulting information on protein higher-order structure. Methods such as electron capture/transfer dissociation (ECD and ETD, or ExD) and ultraviolet photodissociation (UVPD) can produce product ions that are sensitive to structural features of protein complexes. For multi-subunit complexes, a long-held belief is that collisionally activated dissociation (CAD) induces unfolding and release of a subunit, and thus is not useful for higher-order structure characterization. Here we show not only that sequence information can be obtained directly from CAD of native protein complexes but that the fragmentation pattern can deliver higher-order structural information about their gas- and solution-phase structures. Moreover, CAD-generated internal fragments (i.e., fragments containing neither N-/C-termini) reveal structural aspects of protein complexes.

Protein misfolding and aggregation are hallmarks of many severe neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's disease. As a supramolecular ligand that binds to lysine and arginine residues, the molecular tweezer CLR01 was found to modify the aggregation pathway of disease-relevant proteins in vitro and in vivo with beneficial effects on toxicity. However, the molecular mechanisms of how tweezers exert these effects remain mainly unknown, hampering further drug development. Here, we investigate the modulation mechanism of unfolding and aggregation pathways of SOD1, which are involved in amyotrophic lateral sclerosis (ALS), by CLR01. Using a truncated version of the wildtype SOD1 protein, SOD1bar , we show that CLR01 acts on the first step of the aggregation pathway, the unfolding of the SOD1 monomer. CLR01 increases, by ∼10 °C, the melting temperatures of the A4V and G41D SOD1 mutants, which are commonly observed mutations in familial ALS. Molecular dynamics simulations and binding free energy calculations as well as native mass spectrometry and mutational studies allowed us to identify K61 and K92 as binding sites for the tweezers to mediate the stability increase. The data suggest that the modulation of SOD1 conformational stability is a promising target for future developments of supramolecular ligands against neurodegenerative diseases.

Frontotemporal lobar degeneration (FTLD) is the third most common neurodegenerative condition after Alzheimer's and Parkinson's diseases1. FTLD typically presents in 45 to 64 year olds with behavioural changes or progressive decline of language skills2. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions with TAR DNA-binding protein (TDP-43) immunoreactivity3. Here we extracted amyloid fibrils from brains of four patients representing four of the five FTLD-TDP subclasses, and determined their structures by cryo-electron microscopy. Unexpectedly, all amyloid fibrils examined were composed of a 135-residue carboxy-terminal fragment of transmembrane protein 106B (TMEM106B), a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP4. In addition to TMEM106B fibrils, we detected abundant non-fibrillar aggregated TDP-43 by immunogold labelling. Our observations confirm that FTLD-TDP is associated with amyloid fibrils, and that the fibrils are formed by TMEM106B rather than TDP-43.

Different tauopathies are characterized by the isoform-specific composition of the aggregates found in the brain and by structurally distinct tau strains. Although tau oligomers have been implicated as important neurotoxic species, little is known about how the primary structures of the six human tau isoforms affect tau oligomerization because the oligomers are metastable and difficult to analyze. To address this knowledge gap, here, we analyzed the initial oligomers formed by the six tau isoforms in the absence of posttranslational modifications or other manipulations using dot blots probed by an oligomer-specific antibody, native-PAGE/western blots, photo-induced cross-linking of unmodified proteins, mass-spectrometry, and ion-mobility spectroscopy. We found that under these conditions, three-repeat (3R) isoforms are more prone than four-repeat (4R) isoforms to form oligomers. We also tested whether known inhibitors of tau aggregation affect its oligomerization using three small molecules representing different classes of tau aggregation inhibitors, Methylene Blue (MB), the molecular tweezer CLR01, and the all-D peptide TLKIVW, for their ability to inhibit or modulate the oligomerization of the six tau isoforms. Unlike their reported inhibitory effect on tau fibrillation, the inhibitors had little or no effect on the initial oligomerization. Our study provides novel insight into the primary-quaternary structure relationship of human tau and suggests that 3R-tau oligomers may be an important target for future development of compounds targeting pathological tau assemblies.

Top-down mass spectrometry (TD-MS) generates fragment ions that returns information on the polypeptide amino acid sequence. In addition to terminal fragments, internal fragments that result from multiple cleavage events can also be formed. Traditionally, internal fragments are largely ignored due to a lack of available software to reliably assign them, mainly caused by a poor understanding of their formation mechanism. To accurately assign internal fragments, their formation process needs to be better understood. Here, we applied a statistical method to compare fragmentation patterns of internal and terminal fragments of peptides and proteins generated by collisionally activated dissociation (CAD). Internal fragments share similar fragmentation propensities with terminal fragments (e.g., enhanced cleavages N-terminal to proline and C-terminal to acidic residues), suggesting that their formation follows conventional CAD pathways. Internal fragments should be generated by subsequent cleavages of terminal fragments and their formation can be explained by the well-known mobile proton model. In addition, internal fragments can be coupled with terminal fragments to form complementary product ions that span the entire protein sequence. These enhance our understanding of internal fragment formation and can help improve sequencing algorithms to accurately assign internal fragments, which will ultimately lead to more efficient and comprehensive TD-MS analysis of proteins and proteoforms.

Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and β-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.

Syntrophomonas wolfei is an anaerobic syntrophic microbe that degrades short-chain fatty acids to acetate, hydrogen, and/or formate. This thermodynamically unfavorable process proceeds through a series of reactive acyl-Coenzyme A species (RACS). In other prokaryotic and eukaryotic systems, the production of intrinsically reactive metabolites correlates with acyl-lysine modifications, which have been shown to play a significant role in metabolic processes. Analogous studies with syntrophic bacteria, however, are relatively unexplored and we hypothesized that highly abundant acylations could exist in S. wolfei proteins, corresponding to the RACS derived from degrading fatty acids. Here, by mass spectrometry-based proteomics (LC-MS/MS), we characterize and compare acylome profiles of two S. wolfei subspecies grown on different carbon substrates. Because modified S. wolfei proteins are sufficiently abundant to analyze post-translational modifications (PTMs) without antibody enrichment, we could identify types of acylations comprehensively, observing six types (acetyl-, butyryl-, 3-hydroxybutyryl-, crotonyl-, valeryl-, and hexanyl-lysine), two of which have not been reported in any system previously. All of the acyl-PTMs identified correspond directly to RACS in fatty acid degradation pathways. A total of 369 sites of modification were identified on 237 proteins. Structural studies and in vitro acylation assays of a heavily modified enzyme, acetyl-CoA transferase, provided insight on the potential impact of these acyl-protein modifications. The extensive changes in acylation-type, abundance, and modification sites with carbon substrate suggest that protein acylation by RACS may be an important regulator of syntrophy.

Proteins are the primary effectors of function in biology, and thus, complete knowledge of their structure and properties is fundamental to deciphering function in basic and translational research. The chemical diversity of proteins is expressed in their many proteoforms, which result from combinations of genetic polymorphisms, RNA splice variants, and posttranslational modifications. This knowledge is foundational for the biological complexes and networks that control biology yet remains largely unknown. We propose here an ambitious initiative to define the human proteome, that is, to generate a definitive reference set of the proteoforms produced from the genome. Several examples of the power and importance of proteoform-level knowledge in disease-based research are presented along with a call for improved technologies in a two-pronged strategy to the Human Proteoform Project.

Top-down mass spectrometry (TD-MS) of intact proteins results in fragment ions that can be correlated to the protein primary sequence. Fragments generated can either be terminal fragments that contain the N- or C-terminus or internal fragments that contain neither termini. Traditionally in TD-MS experiments, the generation of internal fragments has been avoided because of ambiguity in assigning these fragments. Here, we demonstrate that in TD-MS experiments internal fragments can be formed and assigned in collision-based, electron-based, and photon-based fragmentation methods and are rich with sequence information, allowing for a greater extent of the primary protein sequence to be explained. For the three test proteins cytochrome c, myoglobin, and carbonic anhydrase II, the inclusion of internal fragments in the analysis resulted in approximately 15-20% more sequence coverage, with no less than 85% sequence coverage obtained. Combining terminal fragment and internal fragment assignments results in near complete protein sequence coverage. Hence, by including both terminal and internal fragment assignments in TD-MS analysis, deep protein sequence analysis, allowing for the localization of modification sites more reliably, can be possible.

Top-down mass spectrometry (TD-MS) of peptides and proteins results in product ions that can be correlated to polypeptide sequence. Fragments can either be terminal fragments, which contain either the N- or the C-terminus, or internal fragments that contain neither termini. Normally, only terminal fragments are assigned due to the computational difficulties of assigning internal fragments. Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complementary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum. Data are available via the MassIVE community resource with the identifiers MSV000086788 and MSV000086789.

Acyl modifications vary greatly in terms of elemental composition and site of protein modification. Developing methods to identify acyl modifications more confidently can help to assess the scope of these modifications in large proteomic datasets. The utility of acyl-lysine immonium ions is analyzed for identifying the modifications in proteomic datasets. It is demonstrated that the cyclized immonium ion is a strong indicator of acyl-lysine presence when its rank or relative abundance compared to other ions within a spectrum is considered. Utilizing a stepped collision energy method in a shotgun experiment highlights the immonium ion. By implementing an analysis that accounted for features within each MS2 spectrum, the method clearly identifies peptides with short chain acyl-lysine modifications from complex lysates. Immonium ions can also be used to validate novel acyl modifications; in this study, the first examples of 3-hydroxylpimelyl-lysine modifications are reported and they are validated using immonium ions. Overall these results solidify the use of the immonium ion as a marker for acyl-lysine modifications in complex proteomic datasets.

In biology, it can be argued that if the genome contains the script for a cell's life cycle, then the proteome constitutes an ensemble cast of actors that brings these instructions to life. Their interactions with each other, co-factors, ligands, substrates, and so on, are key to understanding nearly any biological process. Mass spectrometry is well established as the method of choice to determine protein primary structure and location of post-translational modifications. In recent years, top-down fragmentation of intact proteins has been increasingly combined with ionisation of noncovalent assemblies under non-denaturing conditions, i.e., native mass spectrometry. Sequence, post-translational modifications, ligand/metal binding, protein folding, and complex stoichiometry can thus all be probed directly. Here, we review recent developments in this new and exciting field of research. While this work is written primarily from a mass spectrometry perspective, it is targeted to all bioanalytical scientists who are interested in applying these methods to their own biochemistry and chemical biology research.

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation and electron transfer dissociation are widely used for these types of analyses. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (>20 eV) has been suggested to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here, we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that include neither the C-terminus nor the N-terminus of the protein. Conventionally, internal fragments have been disregarded, as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ∼20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ∼50 to ∼99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. When searching for internal fragments during data analysis, previously unassigned peaks can be readily and accurately assigned to confirm a given protein sequence and to enhance the utility of top-down protein sequencing experiments.

Site-specifically modified protein bioconjugates have important applications in biology, chemistry, and medicine. Functionalizing specific protein side chains with enzymes using mild reaction conditions is of significant interest, but remains challenging. Recently, the lysine-isopeptide bond forming activity of the sortase enzyme that builds surface pili in Corynebacterium diphtheriae (CdSrtA) has been reconstituted in vitro. A mutationally activated form of CdSrtA was shown to be a promising bioconjugating enzyme that can attach Leu-Pro-Leu-Thr-Gly peptide fluorophores to a specific lysine residue within the N-terminal domain of the SpaA protein (NSpaA), enabling the labeling of target proteins that are fused to NSpaA. Here we present a detailed analysis of the CdSrtA catalyzed protein labeling reaction. We show that the first step in catalysis is rate limiting, which is the formation of the CdSrtA-peptide thioacyl intermediate that subsequently reacts with a lysine ε-amine in NSpaA. This intermediate is surprisingly stable, limiting spurious proteolysis of the peptide substrate. We report the discovery of a new enzyme variant (CdSrtAΔ) that has significantly improved transpeptidation activity, because it completely lacks an inhibitory polypeptide appendage ("lid") that normally masks the active site. We show that the presence of the lid primarily impairs formation of the thioacyl intermediate and not the recognition of the NSpaA substrate. Quantitative measurements reveal that CdSrtAΔ generates its cross-linked product with a catalytic turnover number of 1.4 ± 0.004 h-1 and that it has apparent KM values of 0.16 ± 0.04 and 1.6 ± 0.3 mM for its NSpaA and peptide substrates, respectively. CdSrtAΔ is 7-fold more active than previously studied variants, labeling >90% of NSpaA with peptide within 6 h. The results of this study further improve the utility of CdSrtA as a protein labeling tool and provide insight into the enzyme catalyzed reaction that underpins protein labeling and pilus biogenesis.

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m/z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m/z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This report serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m/z.

Studies have successfully elucidated the mechanism of action of several effector domains that comprise the multifunctional-autoprocessing repeats-in-toxins (MARTX) toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy (MCF)-like effector and its cellular effects remains unknown. In this study, we identify the host cell factors that activate in vivo and in vitro MCF autoprocessing as adenosine diphosphate (ADP)-Ribosylation Factor 1 (ARF1) and ADP-Ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active [guanosine triphosphate (GTP)-bound] form compared to the inactive [guanosine diphosphate (GDP)-bound] form. Subsequent to auto-cleavage, MCF is acetylated on its exposed N-terminal glycine residue. Acetylation apparently does not dictate subcellular localization as MCF is found localized throughout the cell. However, the cleaved form of MCF gains the ability to bind to the specialized lipid phosphatidylinositol 5-phosphate enriched in Golgi and other membranes necessary for endocytic trafficking, suggesting that a fraction of MCF may be subcellularly localized. Traditional thin-section electron microscopy, high-resolution cryoAPEX localization, and fluorescent microscopy show that MCF causes Golgi dispersal resulting in extensive vesiculation. In addition, host mitochondria are disrupted and fragmented. Mass spectrometry analysis found no reproducible modifications of ARF1 suggesting that ARF1 is not post-translationally modified by MCF. Further, catalytically active MCF does not stably associate with ARF1. Our data indicate not only that ARF1 is a cross-kingdom activator of MCF, but also that MCF may mediate cytotoxicity by directly targeting another yet to be identified protein. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression.

PurposePotential acute exposure to ionizing radiation in nuclear or radiological accidents presents complex mass casualty scenarios that demand prompt triage and treatment decisions. Due to delayed symptoms and varied response of radiation victims, there is an urgent need to develop robust biomarkers to assess the extent of injuries in individuals.Experimental designThe transcription factor Nrf2 is the master of redox homeostasis and there is transcriptional evidence of Nrf2-dependent antioxidant response activation upon radiation. The biomarker potential of Nrf2-dependent downstream target enzymes is investigated by measuring their response in bone marrow extracted from C57Bl/6 and C3H mice of both genders for up to 4 days following 6 Gy total body irradiation using targeted MS.ResultsOverall, C57Bl/6 mice have a stronger proteomic response than C3H mice. In both strains, male mice have more occurrences of upregulation in antioxidant enzymes than female mice. For C57Bl/6 male mice, three proteins show elevated abundances after radiation exposure: catalase, superoxide dismutase 1, and heme oxygenase 1. Across both strains and genders, glutathione S-transferase Mu 1 is consistently decreased.Conclusions and clinical relevanceThis study provides the basis for future development of organ-specific protein biomarkers used in diagnostic blood test for radiation injury.

Electrospray ionization mass spectrometry (ESI-MS) is a ubiquitously used analytical method applied across multiple departments in biopharma, ranging from early research discovery to process development. Accurate, efficient, and consistent protein MS spectral deconvolution across multiple instrument and detector platforms (time-of-flight, Orbitrap, Fourier-transform ion cyclotron resonance) is essential. When proteins are ionized during the ESI process, a distribution of consecutive multiply charged ions are observed on the m/z scale, either positive [M + nH]n+ or negative [M - nH]n- depending on the ionization polarity. The manual calculation of the neutral molecular weight (MW) of single proteins measured by ESI-MS is simple; however, algorithmic deconvolution is required for more complex protein mixtures to derive accurate MWs. Multiple deconvolution algorithms have evolved over the past two decades, all of which have their advantages and disadvantages, in terms of speed, user-input parameters (or ideally lack thereof), and whether they perform optimally on proteins analyzed under denatured or native-MS and solution conditions. Herein, we describe the utility of a parsimonious deconvolution algorithm (explaining the observed spectra with a minimum number of masses) to process a wide range of highly diverse biopharma relevant and research grade proteins and complexes (PEG-GCSF; an IgG1k; IgG1- and IgG2-biotin covalent conjugates; the membrane protein complex AqpZ; a highly polydisperse empty MSP1D1 nanodisc and the tetradecameric chaperone protein complex GroEL) analyzed under native-MS, denaturing LC-MS, and positive and negative modes of ionization, using multiple instruments and therefore multiple data formats. The implementation of a comb filter and peak sharpening option is also demonstrated to be highly effective for deconvolution of highly polydisperse and enhanced separation of a low level lysine glycation post-translational modification (+162.1 Da), partially processed heavy chain lysine residues (+128.1 Da), and loss of N-acetylglucosamine (GlcNAc; -203.1 Da).

Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides 52LFRTVLDSGISEVR65 and 89VQDDFVEIH98, identified within this study, with the l-isoaspartate modification introduced at Asp58 and Asp91, respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native 52LFRTVLDSGISEVR65 peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin 66SDRDKFVIFL(isoAsp)VKHF80, results in enhanced amyloid formation in vitro The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of 13 C-atoms from 1-[13 C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.

Mutations in superoxide dismutase 1 (SOD1) cause 15-20% of familial amyotrophic lateral sclerosis (fALS) cases. The resulting amino acid substitutions destabilize SOD1s protein structure, leading to its self-assembly into neurotoxic oligomers and aggregates, a process hypothesized to cause the characteristic motor-neuron degeneration in affected individuals. Currently, effective disease-modifying therapy is not available for ALS. Molecular tweezers prevent formation of toxic protein assemblies, yet their protective action has not been tested previously on SOD1 or in the context of ALS. Here, we tested the molecular tweezer CLR01-a broad-spectrum inhibitor of the self-assembly and toxicity of amyloid proteins-as a potential therapeutic agent for ALS. Using recombinant WT and mutant SOD1, we found that CLR01 inhibited the aggregation of all tested SOD1 forms in vitro Next, we examined whether CLR01 could prevent the formation of misfolded SOD1 in the G93A-SOD1 mouse model of ALS and whether such inhibition would have a beneficial therapeutic effect. CLR01 treatment decreased misfolded SOD1 in the spinal cord significantly. However, these histological findings did not correlate with improvement of the disease phenotype. A small, dose-dependent decrease in disease duration was found in CLR01-treated mice, relative to vehicle-treated animals, yet motor function did not improve in any of the treatment groups. These results demonstrate that CLR01 can inhibit SOD1 misfolding and aggregation both in vitro and in vivo, but raise the question whether such inhibition is sufficient for achieving a therapeutic effect. Additional studies in other less aggressive ALS models may be needed to determine the therapeutic potential of this approach.

Mutations in superoxide dismutase 1 (SOD1) cause 15-20% of familial amyotrophic lateral sclerosis (fALS) cases. The resulting amino acid substitutions destabilize SOD1's protein structure, leading to its self-assembly into neurotoxic oligomers and aggregates, a process hypothesized to cause the characteristic motor-neuron degeneration in affected individuals. Currently, effective disease-modifying therapy is not available for ALS. Molecular tweezers prevent formation of toxic protein assemblies, yet their protective action has not been tested previously on SOD1 or in the context of ALS. Here, we tested the molecular tweezer CLR01-a broad-spectrum inhibitor of the self-assembly and toxicity of amyloid proteins-as a potential therapeutic agent for ALS. Using recombinant WT and mutant SOD1, we found that CLR01 inhibited the aggregation of all tested SOD1 forms in vitro Next, we examined whether CLR01 could prevent the formation of misfolded SOD1 in the G93A-SOD1 mouse model of ALS and whether such inhibition would have a beneficial therapeutic effect. CLR01 treatment decreased misfolded SOD1 in the spinal cord significantly. However, these histological findings did not correlate with improvement of the disease phenotype. A small, dose-dependent decrease in disease duration was found in CLR01-treated mice, relative to vehicle-treated animals, yet motor function did not improve in any of the treatment groups. These results demonstrate that CLR01 can inhibit SOD1 misfolding and aggregation both in vitro and in vivo, but raise the question whether such inhibition is sufficient for achieving a therapeutic effect. Additional studies in other less aggressive ALS models may be needed to determine the therapeutic potential of this approach.

Native top-down mass spectrometry (MS) and ion mobility spectrometry (IMS) were applied to characterize the interaction of a molecular tweezer assembly modulator, CLR01, with tau, a protein believed to be involved in a number of neurodegenerative disorders, including Alzheimer's disease. The tweezer CLR01 has been shown to inhibit aggregation of amyloidogenic polypeptides without toxic side effects. ESI-MS spectra for different forms of tau protein (full-length, fragments, phosphorylated, etc.) in the presence of CLR01 indicate a primary binding stoichiometry of 1:1. The relatively high charging of the protein measured from non-denaturing solutions is typical of intrinsically disordered proteins, such as tau. Top-down mass spectrometry using electron capture dissociation (ECD) is a tool used to determine not only the sites of post-translational modifications but also the binding site(s) of non-covalent interacting ligands to biomolecules. The intact protein and the protein-modulator complex were subjected to ECD-MS to obtain sequence information, map phosphorylation sites, and pinpoint the sites of inhibitor binding. The ESI-MS study of intact tau proteins indicates that top-down MS is amenable to the study of various tau isoforms and their post-translational modifications (PTMs). The ECD-MS data point to a CLR01 binding site in the microtubule-binding region of tau, spanning residues K294-K331, which includes a six-residue nucleating segment PHF6 (VQIVYK) implicated in aggregation. Furthermore, ion mobility experiments on the tau fragment in the presence of CLR01 and phosphorylated tau reveal a shift towards a more compact structure. The mass spectrometry study suggests a picture for the molecular mechanism of the modulation of protein-protein interactions in tau by CLR01. Graphical Abstract ᅟ.

Therapeutic target characterization involves many components, including accurate molecular weight (MW) determination. Knowledge of the accurate MW allows one to detect the presence of post-translational modifications, proteolytic cleavages, and importantly, if the correct construct has been generated and purified. Denaturing liquid chromatography-mass spectrometry (LC-MS) can be an attractive method for obtaining this information. However, membrane protein LC-MS methodology has remained relatively under-explored and under-incorporated in comparison to methods for soluble proteins. Here, systematic investigation of multiple gradients and column chemistries has led to the development of a 5 min denaturing LC-MS method for acquiring membrane protein accurate MW measurements. Conditions were interrogated with membrane proteins, such as GPCRs and ion channels, as well as bispecific antibody constructs of variable sizes with the aim to provide the community with rapid LC-MS methods necessary to obtain chromatographic and accurate MW measurements in a medium- to high-throughput manner. The 5 min method detailed has successfully produced MW measurements for hydrophobic proteins with a wide MW range (17.5 to 105.3 kDa) and provided evidence that some constructs indeed contain unexpected modifications or sequence clipping. This rapid LC-MS method is also capable of baseline separating formylated and nonformylated aquaporinZ membrane protein.

Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass‐sensing RsgI‐type anti‐σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti‐σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/β/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn‐Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti‐σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism. [ABSTRACT FROM AUTHOR]

Native mass spectra of large, polydisperse biomolecules with repeated subunits, such as lipoprotein Nanodiscs, can often be challenging to analyze by conventional methods. The presence of tens of closely spaced, overlapping peaks in these mass spectra can make charge state, total mass, or subunit mass determinations difficult to measure by traditional methods. Recently, we introduced a Fourier Transform-based algorithm that can be used to deconvolve highly congested mass spectra for polydisperse ion populations with repeated subunits and facilitate identification of the charge states, subunit mass, charge-state-specific, and total mass distributions present in the ion population. Here, we extend this method by investigating the advantages of using overtone peaks in the Fourier spectrum, particularly for mass spectra with low signal-to-noise and poor resolution. This method is illustrated for lipoprotein Nanodisc mass spectra acquired on three common platforms, including the first reported native mass spectrum of empty "large" Nanodiscs assembled with MSP1E3D1 and over 300 noncovalently associated lipids. It is shown that overtone peaks contain nearly identical stoichiometry and charge state information to fundamental peaks but can be significantly better resolved, resulting in more reliable reconstruction of charge-state-specific mass spectra and peak width characterization. We further demonstrate how these parameters can be used to improve results from Bayesian spectral fitting algorithms, such as UniDec. Graphical Abstract ᅟ.

Structural characterization of intrinsically disordered proteins (IDPs) has been a major challenge in the field of protein science due to limited capabilities to obtain full-length high-resolution structures. Native ESI-MS with top-down MS was utilized to obtain structural features of protein-ligand binding for the Parkinson's disease-related protein, α-synuclein (αSyn), which is natively unstructured. Binding of heavy metals has been implicated in the accelerated formation of αSyn aggregation. Using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, native top-down MS with various fragmentation methods, including electron capture dissociation (ECD), collisional activated dissociation (CAD), and multistage tandem MS (MS3), deduced the binding sites of cobalt and manganese to the C-terminal region of the protein. Ion mobility MS (IM-MS) revealed a collapse toward compacted states of αSyn upon metal binding. The combination of native top-down MS and IM-MS provides structural information of protein-ligand interactions for intrinsically disordered proteins. Graphical Abstract ᅟ.

Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (CdSrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created CdSrtA3M, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. CdSrtA3M mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that CdSrtA3M can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2M synthesizes pilus fibers with correct Lys-Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA-SrtA-SpaA polymerization intermediate depicts SrtA2M sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.

Trifluoroacetic acid (TFA) is often used as a mobile phase modifier to enhance reversed phase chromatographic performance. TFA adjusts solution pH and is an ion-pairing agent, but it is not typically suitable for electrospray ionization-mass spectrometry (ESI-MS) and liquid chromatography/MS (LC/MS) because of its significant signal suppression. Supercharging agents elevate peptide and protein charge states in ESI, increasing tandem MS (MS/MS) efficiency. Here, LC/MS protein supercharging was effected by adding agents to LC mobile phase solvents. Significantly, the ionization suppression generally observed with TFA was, for the most part, rescued by supercharging agents, with improved separation efficiency (higher number of theoretical plates) and lowered detection limits.

The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard.