RESUMO
Dysregulated glycerophospholipid (GP) metabolism in the brain is associated with the progression of neurodegenerative diseases including Alzheimer's disease (AD). Routine liquid chromatography-mass spectrometry (LC-MS)-based large-scale lipidomic methods often fail to elucidate subtle yet important structural features such as sn-position, hindering the precise interrogation of GP molecules. Leveraging high-resolution demultiplexing (HRdm) ion mobility spectrometry (IMS), we develop a four-dimensional (4D) lipidomic strategy to resolve GP sn-position isomers. We further construct a comprehensive experimental 4D GP database of 498 GPs identified from the mouse brain and an in-depth extended 4D library of 2500 GPs predicted by machine learning, enabling automated profiling of GPs with detailed acyl chain sn-position assignment. Analyzing three mouse brain regions (hippocampus, cerebellum, and cortex), we successfully identify a total of 592 GPs including 130 pairs of sn-position isomers. Further temporal GPs analysis in the three functional brain regions illustrates their metabolic alterations in AD progression.
Assuntos
Doença de Alzheimer , Encéfalo , Glicerofosfolipídeos , Lipidômica , Animais , Doença de Alzheimer/metabolismo , Lipidômica/métodos , Glicerofosfolipídeos/metabolismo , Camundongos , Encéfalo/metabolismo , Espectrometria de Mobilidade Iônica/métodos , Masculino , Cerebelo/metabolismo , Modelos Animais de Doenças , Cromatografia Líquida/métodos , Camundongos Endogâmicos C57BL , Hipocampo/metabolismoRESUMO
Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.
Assuntos
Metano , Metano/análogos & derivados , Metano/química , Humanos , Pegadas de Proteínas/métodos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ligação Proteica , Espectrometria de MassasRESUMO
The American lobster, Homarus americanus, is not only of considerable economic importance but has also emerged as a premier model organism in neuroscience research. Neuropeptides, an important class of cell-to-cell signaling molecules, play crucial roles in a wide array of physiological and psychological processes. Leveraging the recently sequenced high-quality draft genome of the American lobster, our study sought to profile the neuropeptidome of this model organism. Employing advanced mass spectrometry techniques, we identified 24 neuropeptide precursors and 101 unique mature neuropeptides in Homarus americanus. Intriguingly, 67 of these neuropeptides were discovered for the first time. Our findings provide a comprehensive overview of the peptidomic attributes of the lobster's nervous system and highlight the tissue-specific distribution of these neuropeptides. Collectively, this research not only enriches our understanding of the neuronal complexities of the American lobster but also lays a foundation for future investigations into the functional roles that these peptides play in crustacean species. The mass spectrometry data have been deposited in the PRIDE repository with the identifier PXD047230.
Assuntos
Sequência de Aminoácidos , Nephropidae , Neuropeptídeos , Proteômica , Animais , Nephropidae/metabolismo , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/análise , Proteômica/métodos , Espectrometria de Massas , Dados de Sequência MolecularRESUMO
Amino acids (AAs) in the d-form are involved in multiple pivotal neurological processes, although their l-enantiomers are most commonly found. Mass spectrometry-based analysis of low-abundance d-AAs has been hindered by challenging enantiomeric separation from l-AAs, low sensitivity for detection, and lack of suitable internal standards for accurate quantification. To address these critical gaps, N,N-dimethyl-l-leucine (l-DiLeu) tags are first validated as novel chiral derivatization reagents for chromatographic separation of 20 pairs of d/l-AAs, allowing the construction of a 4-plex isobaric labeling strategy for enantiomer-resolved quantification through single step tagging. Additionally, the creative design of N,N-dimethyl-d-leucine (d-DiLeu) reagents offers an alternative approach to generate analytically equivalent internal references of d-AAs using d-DiLeu-labeled l-AAs. By labeling cost-effective l-AA standards using paired d- and l-DiLeu, this approach not only enables absolute quantitation of both d-AAs and l-AAs from complex biological matrices with enhanced precision but also significantly boosts the combined signal intensities from all isobaric channels, greatly improving the detection and quantitation of low-abundance AAs, particularly d-AAs. We term this quantitative strategy CHRISTMAS, which stands for chiral pair isobaric labeling strategy for multiplexed absolute quantitation. Leveraging the ion mobility collision cross section (CCS) alignment, interferences from coeluting isomers/isobars are effectively filtered out to provide improved quantitative accuracy. From wild-type and Alzheimer's disease (AD) mouse brains, we successfully quantified 20 l-AAs and 5 d-AAs. The significant presence and differential trends of certain d-AAs compared to those of their l-counterparts provide valuable insights into the involvement of d-AAs in aging, AD progression, and neurodegeneration.
Assuntos
Aminoácidos , Proteômica , Animais , Camundongos , Aminoácidos/análise , Proteômica/métodos , Leucina/química , Aminas , Cromatografia Líquida/métodosRESUMO
Traumatic stress triggers or exacerbates multiple psychiatric illnesses, including post-traumatic stress disorder (PTSD). Nevertheless, the neurophysiological mechanisms underlying stress-induced pathology remain unclear, in part due to the limited understanding of neuronal signaling molecules, such as neuropeptides, in this process. Here, we developed mass spectrometry (MS)-based qualitative and quantitative analytical strategies to profile neuropeptides in rats exposed to predator odor (an ethologically relevant analogue of trauma-like stress) versus control subjects (no odor) to determine peptidomic alterations induced by trauma. In total, 628 unique neuropeptides were identified across 5 fear-circuitry-related brain regions. Brain-region-specific changes of several neuropeptide families, including granin, ProSAAS, opioids, cholecystokinin, and tachykinin, were also observed in the stressed group. Neuropeptides from the same protein precursor were found to vary across different brain regions, indicating the site-specific effects of predator stress. This study reveals for the first time the interaction between neuropeptides and traumatic stress, providing insights into the molecular mechanisms of stress-induced psychopathology and suggesting putative novel therapeutic strategies for disorders such as PTSD.
Assuntos
Neuropeptídeos , Transtornos de Estresse Pós-Traumáticos , Ratos , Animais , Transtornos de Estresse Pós-Traumáticos/metabolismo , Transtornos de Estresse Pós-Traumáticos/psicologia , Encéfalo/metabolismo , Neuropeptídeos/metabolismoRESUMO
Growing evidence supports the confident association between distinct amyloid beta (Aß) isoforms and Alzheimer's Disease (AD) pathogenesis. As such, critical investigations seeking to uncover the translational factors contributing to Aß toxicity represent a venture of significant value. Herein, we comprehensively assess full-length Aß42 stereochemistry, with a specific focus on models that consider naturally-occurring isomerization of Asp and Ser residues. We customize various forms of d-isomerized Aß as natural mimics, ranging from fragments containing a single d residue to full length Aß42 that includes multiple isomerized residues, systematically evaluating their cytotoxicity against a neuronal cell line. Combining multidimensional ion mobility-mass spectrometry experimental data with replica exchange molecular dynamics simulations, we confirm that co-d-epimerization at Asp and Ser residues within Aß42 in both N-terminal and core regions effectively reduces its cytotoxicity. We provide evidence that this rescuing effect is associated with the differential and domain-specific compaction and remodeling of Aß42 secondary structure.
RESUMO
The knowledge of ligand-protein interactions is essential for understanding fundamental biological processes and for the rational design of drugs that target such processes. Carbene footprinting efficiently labels proteinaceous residues and has been used with mass spectrometry (MS) to map ligand-protein interactions. Nevertheless, previous footprinting studies are typically performed at the residue level, and therefore, the resolution may not be high enough to couple with conventional crystallography techniques. Herein we developed a subresidue footprinting strategy based on the discovery that carbene labeling produces subresidue peptide isomers and the intensity changes of these isomers in response to ligand binding can be exploited to delineate ligand-protein topography at the subresidue level. The established workflow combines carbene footprinting, extended liquid chromatographic separation, and ion mobility (IM)-MS for efficient separation and identification of subresidue isomers. Analysis of representative subresidue isomers located within the binding cleft of lysozyme and those produced from an amyloid-ß segment have both uncovered structural information heretofore unavailable by residue-level footprinting. Lastly, a "real-world" application shows that the reactivity changes of subresidue isomers at Phe399 can identify the interactive nuances between estrogen-related receptor α, a potential drug target for cancer and metabolic diseases, with its three ligands. These findings have significant implications for drug design. Taken together, we envision the subresidue-level resolution enabled by IM-MS-coupled carbene footprinting can bridge the gap between structural MS and the more-established biophysical tools and ultimately facilitate diverse applications for fundamental research and pharmaceutical development.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Metano/análogos & derivados , Muramidase/metabolismo , Receptores de Estrogênio/metabolismo , Peptídeos beta-Amiloides/química , Animais , Sítios de Ligação , Galinhas , Humanos , Ligantes , Metano/química , Muramidase/química , Ligação Proteica , Receptores de Estrogênio/química , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Lysine-conjugated antibody-drug conjugates (ADCs) are formed by attaching cytotoxic drugs to reactive lysine residues of monoclonal antibodies (mAbs) through chemical linkers. During production, the payloads are conjugated nonspecifically to lysine residues in mAbs, resulting in a heterogeneous mixture of ADCs with both different number and conjugation sites of drug payloads per mAb. On account of the drug conjugation sites and levels that both have significant influences on physical and pharmaceutical properties of ADCs, a reliable and straightforward approach for conjugation site analysis for ADCs is highly demanded. Herein, we used a lysine-conjugated ADC, Trastuzumab-MCC-DM1 (T-DM1), as a model ADC, and described an integrative strategy that combines the signature ion fingerprinting method for rapid and reliable filtering of DM1-conjugated peptides, and the normalized area quantitation approach for accurately gauging the conjugation levels for each identified site. This approach is believed to be readily applicable to other maytansinoid derivatives-modified ADCs, and more importantly, universally applicable to lysine-conjugated ADCs for both the recognition of conjugation sites and the measurement of conjugation levels.
Assuntos
Antineoplásicos Imunológicos/análise , Antineoplásicos Imunológicos/química , Imunoconjugados/análise , Imunoconjugados/química , Lisina/química , Cromatografia Líquida , Desenvolvimento de Medicamentos , Humanos , Estrutura Molecular , Peptídeos/química , Espectrometria de Massas em TandemRESUMO
Metronomic chemotherapy, a relatively new dosing paradigm for anticancer therapy, is an alternative to traditional chemotherapy that uses maximal tolerated dose (MTD). Although these two dosing regimens both lead to tumor cell death, how cell metabolism is differentially affected during apoptosis remains elusive. Herein, we employed metabolomics to monitor the metabolic profiles of MCF-7 cells in response to the two dosing regimens that mimic MTD and MN treatments using a model chemotherapeutic drug, doxorubicin (Dox), and correlated the changes of metabolic genes examined by PCR array to integratively describe the reprogrammed metabolic patterns. We found glycolysis, amino acid, and nucleotide synthesis-associated metabolic pathways were activated in response to the MN treatment, whereas these pathways were inhibited in a pronounced way in response to the MTD treatment. Direct supplementation of key metabolites and pharmacological modulation of targeted metabolic enzymes can both regulate cell fates. Subsequently, we tested the combined use of MN dosing with targeted metabolic intervention using a normal cell line and found the combined treatment hardly affected its apoptotic rate. Our in vitro findings using MCF-7 and MCF-10A cells thus suggest the promising perspective of combining MN dosing of chemotherapeutic agents with metabolic modulation to selectively kill cancer cells rather than normal cells.
Assuntos
Administração Metronômica , Neoplasias da Mama/tratamento farmacológico , Proteínas de Neoplasias/metabolismo , Proteoma/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Doxorrubicina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Proteínas de Neoplasias/genética , Proteoma/genéticaRESUMO
In this paper, new expressions of the channel-correlation coefficient and its components (the large- and small-scale channel-correlation coefficients) for a plane wave are derived for a horizontal link in moderate-to-strong non-Kolmogorov turbulence using a generalized effective atmospheric spectrum which includes finite-turbulence inner and outer scales and high-wave-number "bump". The closed-form expression of the average bit error rate (BER) of the coherent free-space optical communication system is derived using the derived channel-correlation coefficients and an α-µ distribution to approximate the sum of the square root of arbitrarily correlated Gamma-Gamma random variables. Analytical results are provided to investigate the channel correlation and evaluate the average BER performance. The validity of the proposed approximation is illustrated by Monte Carlo simulations. This work will help with further investigation of the fading correlation in spatial diversity systems.
RESUMO
Emerging evidence support that temporal dynamics is pivotal for signaling molecules in orchestrating smart responses to diverse stimuli. p53 is such a signaling molecule that employs temporal dynamics for the selective activation of downstream target genes and ultimately for cell fate decision. Yet how this fine-tuned p53 machinery is quantitatively decoded remains largely unclear. Here we report a quantitative mechanism defining how p53 dynamics orchestrates with binding affinity to target genes for cell fate decision. Treating cells with a genotoxic drug doxorubicin at various doses and durations, we found that a mild and prolonged challenge triggered sequential p53 pulses and ultimately resulted in a terminal pulse enacting apoptosis in a comparable rate with that induced by an acute and high-dose treatment. To transactivate proapoptotic genes and thereafter executing apoptosis, p53 must exceed a certain threshold and accumulate for sufficient time at levels above it. Effective cumulative levels above the threshold, defined as E∫p53, but not the total accumulation levels of p53, precisely discriminate survival and apoptotic cells. p53 accumulation below this threshold, even with prolonging time to reach a total level comparable to that from the accumulation over the threshold, could not transactivate proapoptotic genes to which the binding affinity of p53 is lower than that of proarrest genes, and this property is independent of dynamic features. Our findings indicate that the dynamic feature per se does not directly control cell fate, but rather it orchestrates with the binding affinity to target genes to confer an appropriate time window for cell fate choice. Our study provides a quantitative mechanism unifying p53 dynamics and binding affinity to target genes, providing novel insights to understand how p53 can respond quantitatively to chemotherapeutic drugs, and guiding the design of metronomic regimens for chemotherapeutic drugs.
Assuntos
Diferenciação Celular/genética , Proteína Supressora de Tumor p53/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Doxorrubicina/farmacologia , Regulação da Expressão Gênica , Humanos , Células MCF-7 , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Estresse Fisiológico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Endogenous neuropeptides are important signaling molecules that function as regulators of food intake and body weight. Previous work has shown that neuropeptide gene expression levels in a forebrain reward site, the nucleus accumbens (NAc), were changed by feeding. To directly monitor feeding-induced changes in neuropeptide expression levels within the NAc, we employed a combination of cryostat dissection, heat stabilization, neuropeptide extraction and label-free quantitative neuropeptidomics via a liquid chromatography-high resolution mass spectrometry platform. Using this methodology, we described the first neuropeptidome in NAc and discovered that feeding caused the expression level changes of multiple neuropeptides derived from different precursors, especially proSAAS-derived peptides such as Big LEN, PEN and little SAAS. We further investigated the regulatory functions of these neuropeptides derived from the ProSAAS family by performing an intra-NAc microinjection experiment using the identified ProSAAS neuropeptides, 'Big-LEN' and 'PEN'. Big LEN significantly increased rats' food and water intake, whereas both big LEN and PEN affected other behaviors including locomotion, drinking and grooming. In addition, we quantified the feeding-induced changes of peptides from hippocampus, hypothalamus and striatum to reveal the neuropeptide interplay among different anatomical regions. In summary, our study demonstrated neuropeptidomic changes in response to food intake in the rat NAc and other key brain regions. Importantly, the microinfusion of ProSAAS peptides into NAc revealed that they are behaviorally active in this brain site, suggesting the potential use of these peptides as therapeutics for eating disorders.
Assuntos
Ingestão de Alimentos , Neuropeptídeos/análise , Núcleo Accumbens/metabolismo , Proteômica/métodos , Animais , Hipocampo/metabolismo , Hipotálamo/metabolismo , Masculino , Espectrometria de Massas , RatosRESUMO
Apoptosis and senescence are two types of cell fates in response to chemotherapy. Besides canonical pathways that mediate cell fates, cancer cell metabolism has been revealed as a crucial factor affecting cell fate decisions and thus represents a new target for antitumor therapy. Therefore, a comprehensive description of metabolic pathways underlying cell senescence and apoptosis in response to chemotherapy is highly demanded for therapeutic exploitation of both processes. Herein we employed a metabolomics-proteomics combined approach to identify metabolism-associated molecular events that mediate cellular responses to senescence and apoptosis using doxorubicin-treated human breast cancer cells MCF7 as models. Such biomics approach revealed that tricarboxylic acid cycle, pentose phosphate pathway, and nucleotide synthesis pathways were significantly upregulated in the senescent model, whereas fatty acid synthesis was reduced. In apoptotic cells, an overall reduced activity of major metabolic pathways was observed except for the arginine and proline pathway. Combinatorially, these data show the utility of biomics in exploring biochemical mechanism-based differences between apoptosis and senescence and reveal an unprecedented finding of the metabolic events that were induced for survival by facilitating ROS elimination and DNA damage repair in senescent cells, while they were downregulated in apoptotic cells when DNA damage was irreparable.
Assuntos
Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica/métodos , Proteômica/métodos , Ciclo do Ácido Cítrico , Dano ao DNA , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Ácidos Graxos/biossíntese , Humanos , Células MCF-7 , Nucleotídeos/biossíntese , Via de Pentose Fosfato , Espécies Reativas de Oxigênio/metabolismoRESUMO
Trastuzumab-MCC-DM1 (T-DM1) is an antibody-drug conjugate (ADC) that consists of a monoclonal antibody (mAb) trastuzumab non-cleavably linked to a cytotoxic drug DM1. During production, the DM1 agents were conjugated to the lysine residues of the mAb in a non-specific manner, yielding a heterogeneous mixture of ADC molecules that differ with respect to both the number and the conjugation sites of DM1 per mAb molecule. Since drug conjugation sites of ADC can significantly impact properties such as stability and pharmacokinetic behaviors, a rapid and reliable approach for conjugation site analysis of ADCs is highly demanded. Herein, we have employed a signature ion fingerprinting approach to specifically determine lysine residues with DM1 conjugation, and developed a normalized peak area quantitation method to characterize the percentage of DM1-conjugated lysine for each putative site using a T-DM1 biosimilar as a model drug. With this integrative approach, 38 lysine residues were identified with DM1 conjugation among 90 possible sites. More interestingly, we found that the T-DM1 biosimilar exhibited a specific preference of DM1-conjugation for several lysine residues, and such preference was consistent among three production batches. A molecular modeling approach was subsequently utilized to analyze all the conjugation sites, and revealed an intriguing correlation of the conjugated residue's microenvironment with the conjugation level. In summary, our study introduced an approach that is widely applicable to ADCs of interest for conjugation site analysis. Moreover, it suggests the necessity of performing conjugation site analysis for product and process characterization and also for routine use in lot release and stability testing of manufactured ADCs.
Assuntos
Imunoconjugados/química , Maitansina/química , Trastuzumab/química , Cromatografia Líquida , Espectrometria de MassasRESUMO
The characterization of herbal prescriptions serves as a foundation for quality control and regulation of herbal medicines. Previously, the characterization of herbal chemicals from natural medicines often relied on the analysis of signature fragment ions from the acquired tandem mass spectrometry (MS/MS) spectra with prior knowledge of the herbal species present in the herbal prescriptions of interest. Nevertheless, such an approach is often limited to target components, and it risks missing the critical components that we have no prior knowledge of. We previously reported a "diagnostic ion-guided network bridging" strategy. It is a generally applicable and robust approach to analyze unknown substances from complex mixtures in an untargeted manner. In this study, we have developed a standalone software named "Nontargeted Diagnostic Ion Network Analysis (NINA)" with a graphical user interface based on a strategy for post-acquisition data analysis. NINA allows one to rapidly determine the nontargeted diagnostic ions (NIs) by summarizing all of the fragment ions shared by the precursors from the acquired MS/MS spectra. A NI-guided network using bridging components that possess two or more NIs can then be established via NINA. With such a network, we could sequentially identify the structures of all the NIs once a single compound has been identified de novo. The structures of NIs can then be used as "priori" knowledge to narrow the candidates containing the sub-structure of the corresponding NI from the database hits. Subsequently, we applied the NINA software to the characterization of a model herbal prescription, Re-Du-Ning injection, and rapidly identified 56 herbal chemicals from the prescription using an ultra-performance liquid chromatography quadrupole time-of-flight system in the negative mode with no knowledge of the herbal species or herbal chemicals in the mixture. Therefore, we believe the applications of NINA will greatly facilitate the characterization of complex mixtures, such as natural medicines, especially when no advance information is available. In addition to herbal medicines, the NINA-based workflow will also benefit many other fields, such as environmental analysis, nutritional science, and forensic analysis.
Assuntos
Produtos Biológicos/análise , Medicamentos de Ervas Chinesas/análise , Software , Espectrometria de Massas em Tandem/métodos , Software/normas , Espectrometria de Massas em Tandem/normasRESUMO
In the past decades, both the increasing experimental evidence and some results of theoretical investigation on non-Kolmogorov turbulence have been reported. This has prompted the study of optical propagation in non-Kolmogorov atmospheric turbulence. In this paper, based on the thin phase screen model and a non-Kolmogorov power spectrum which owns a generalized power law instead of standard Kolmogorov power law value 11/3 and a generalized amplitude factor instead of constant value 0.033, the temporal power spectrum of irradiance fluctuations for a Gaussian-beam wave is derived in the weak fluctuation regime for a horizontal path. The analytic expressions are obtained and then used to analyze the influence of spectral power law variations on the temporal power spectrum of irradiance fluctuations.