RESUMO
The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community.
RESUMO
Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down mass spectrometry (MS). Improved sequence coverage, critical for reliable annotation of posttranslational modifications and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as LC/MS of several proteins. Experiments were undertaken on multiple instruments, including quadrupole time-of-flight, Orbitrap, and high-field Fourier-transform ion cyclotron resonance (FT-ICR) across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher energy collision dissociation MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical versus even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD or ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions.
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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/ß/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.
Assuntos
Proteínas de Bactérias , Clostridium thermocellum , Simulação de Dinâmica Molecular , Proteólise , Clostridium thermocellum/metabolismo , Clostridium thermocellum/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Fator sigma/química , Fator sigma/metabolismo , Fator sigma/genética , Sequência de Aminoácidos , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Celulossomas/metabolismo , Celulossomas/química , Cristalografia por Raios X , Espectrometria de Massas em Tandem , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genéticaRESUMO
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.
Assuntos
Cisteína , Ouro , Cisteína/química , Ouro/química , Peptídeos/química , Compostos Organoáuricos/química , Compostos Organoáuricos/síntese química , Estrutura MolecularRESUMO
Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are important large biotherapeutics (â¼150 kDa) and high structural complexity that require extensive sequence and structure characterization. Middle-down mass spectrometry (MD-MS) is an emerging technique that sequences and maps subunits larger than those released by trypsinolysis. It avoids potentially introducing artifactual modifications that may occur in bottom-up MS while achieving higher sequence coverage compared to top-down MS. However, returning complete sequence information by MD-MS is still challenging. Here, we show that assigning internal fragments in direct infusion MD-MS of a mAb and an ADC substantially improves their structural characterization. For MD-MS of the reduced NIST mAb, including internal fragments recovers nearly 100% of the sequence by accessing the middle sequence region that is inaccessible by terminal fragments. The identification of important glycosylations can also be improved after the inclusion of internal fragments. For the reduced lysine-linked IgG1-DM1 ADC, we show that considering internal fragments increases the DM1 conjugation sites coverage to 80%, comparable to the reported 83% coverage achieved by peptide mapping on the same ADC (Luo et al. Anal. Chem. 2016, 88, 695-702). This study expands our work on the application of internal fragment assignments in top-down MS of mAbs and ADCs and can be extended to other heterogeneous therapeutic molecules such as multispecifics and fusion proteins for more widespread applications.
Assuntos
Anticorpos Monoclonais , Imunoconjugados , Anticorpos Monoclonais/química , Imunoconjugados/química , Espectrometria de Massas/métodos , Mapeamento de Peptídeos , Lisina/químicaRESUMO
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.
Assuntos
Doença de Alzheimer , Modelos Animais de Doenças , Nanopartículas de Magnetita , Proteínas tau , Animais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Proteínas tau/metabolismo , Proteínas tau/química , Camundongos , Humanos , Nanopartículas de Magnetita/química , Amiloide/metabolismo , Amiloide/química , Camundongos Transgênicos , Comportamento Animal/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Agregação Patológica de Proteínas/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/efeitos dos fármacosRESUMO
Proteoforms, which arise from post-translational modifications, genetic polymorphisms and RNA splice variants, play a pivotal role as drivers in biology. Understanding proteoforms is essential to unravel the intricacies of biological systems and bridge the gap between genotypes and phenotypes. By analysing whole proteins without digestion, top-down proteomics (TDP) provides a holistic view of the proteome and can decipher protein function, uncover disease mechanisms and advance precision medicine. This Primer explores TDP, including the underlying principles, recent advances and an outlook on the future. The experimental section discusses instrumentation, sample preparation, intact protein separation, tandem mass spectrometry techniques and data collection. The results section looks at how to decipher raw data, visualize intact protein spectra and unravel data analysis. Additionally, proteoform identification, characterization and quantification are summarized, alongside approaches for statistical analysis. Various applications are described, including the human proteoform project and biomedical, biopharmaceutical and clinical sciences. These are complemented by discussions on measurement reproducibility, limitations and a forward-looking perspective that outlines areas where the field can advance, including potential future applications.