RESUMO
Over the last two decades, hundreds of new psychoactive substances (NPSs), also known as "designer drugs", have emerged on the illicit drug market. The toxic and potentially fatal effects of these compounds oblige laboratories around the world to screen for NPS in seized materials and biological samples, commonly using high-resolution mass spectrometry. However, unambiguous identification of a NPS by mass spectrometry requires comparison to data from analytical reference materials, acquired on the same instrument. The sheer number of NPSs that are available on the illicit market, and the pace at which new compounds are introduced, means that forensic laboratories must make difficult decisions about which reference materials to acquire. Here, we asked whether retrospective suspect screening of population-scale mass spectrometry data could provide a data-driven platform to prioritize emerging NPSs for assay development. We curated a suspect database of precursor and diagnostic fragment ion masses for 83 emerging NPSs and used this database to retrospectively screen mass spectrometry data from 12,727 urine drug screens from one Canadian province. We developed integrative computational strategies to prioritize the most reliable identifications and tracked the frequency of these identifications over a 3 year study period between August 2019 and August 2022. The resulting data were used to guide the acquisition of new reference materials, which were in turn used to validate a subset of the retrospective identifications. Last, we took advantage of matching clinical reports for all 12,727 samples to systematically benchmark the accuracy of our retrospective data analysis approach. Our work opens up new avenues to enable the rapid detection of emerging illicit drugs through large-scale reanalysis of mass spectrometry data.
Assuntos
Drogas Ilícitas , Psicotrópicos , Estudos Retrospectivos , Psicotrópicos/análise , Canadá , Espectrometria de Massas/métodos , Drogas Ilícitas/análise , Detecção do Abuso de Substâncias/métodosRESUMO
Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of cerebrospinal fluid and serum samples from 111 patients with acute SCI and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at 6 months with an area under the receiver operating characteristic curve of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response and establish the cerebrospinal fluid abundance of glial fibrillary acidic protein as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.
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Proteína Glial Fibrilar Ácida/sangue , Proteína Glial Fibrilar Ácida/líquido cefalorraquidiano , Traumatismos da Medula Espinal/sangue , Traumatismos da Medula Espinal/líquido cefalorraquidiano , Animais , Feminino , Humanos , Proteômica , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , SuínosRESUMO
Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) has emerged as a topic of great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.
Assuntos
Antígenos de Neoplasias/metabolismo , Compostos Bicíclicos com Pontes/química , Inibidores Enzimáticos/química , Histona Acetiltransferases/metabolismo , Hialuronoglucosaminidase/metabolismo , Sequência de Aminoácidos , Encéfalo/metabolismo , Compostos Bicíclicos com Pontes/metabolismo , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/metabolismo , Histona Acetiltransferases/antagonistas & inibidores , Humanos , Hialuronoglucosaminidase/antagonistas & inibidores , Espectrometria de Massas , Peptídeos/análise , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Relação Estrutura-Atividade , Tiazolidinas/química , Tiazolidinas/metabolismo , Cadeia alfa da beta-Hexosaminidase/antagonistas & inibidores , Cadeia alfa da beta-Hexosaminidase/metabolismoRESUMO
Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Membrana/genética , Mycobacterium tuberculosis/genética , Tuberculose/genética , Transportadores de Cassetes de Ligação de ATP/química , Citosol/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/ultraestrutura , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/patogenicidade , Mycobacterium tuberculosis/ultraestrutura , Proteínas Nucleares/genética , Fosforilação/genética , Transdução de Sinais/genética , Imagem Individual de Molécula , Tuberculose/microbiologiaRESUMO
Methylation of specific lysine residues in the C terminus of p53 is thought to govern p53-dependent transcription following genotoxic and oncogenic stress. In particular, Set7/9 (KMT7)-mediated monomethylation of human p53 at lysine 372 (p53K372me1) was suggested to be essential for p53 activation in human cell lines. This finding was confirmed in a Set7/9 knockout mouse model (Kurash et al., 2008). In an independent knockout mouse strain deficient in Set7/9, we have investigated its involvement in p53 regulation and find that cells from these mice are normal in their ability to induce p53-dependent transcription following genotoxic and oncogenic insults. Most importantly, we detect no impairment in canonical p53 functions in these mice, indicating that Set7/9-mediated methylation of p53 does not seem to represent a major regulatory event and does not appreciably control p53 activity in vivo.
Assuntos
Proteínas Metiltransferases/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose/genética , Ciclo Celular , Senescência Celular/genética , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Metiltransferases/metabolismo , Proteínas Metiltransferases/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific features.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mucilagem Vegetal/metabolismo , Sementes/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Parede Celular/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Epiderme Vegetal/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Corticosteroid-binding globulin (CBG) was isolated from chicken serum and identified by mass spectrometry and genomic analysis. This revealed that the organization and synteny of avian and mammalian SerpinA6 genes are conserved. Recombinant zebra finch CBG steroid-binding properties reflect those of the natural protein in plasma and confirm its identity. Zebra finch and rat CBG crystal structures in complex with cortisol resemble each other, but their primary structures share only â¼40% identity, and their steroid-binding site topographies differ in several unexpected ways. Remarkably, a tryptophan that anchors ligands in mammalian CBG steroid-binding sites is replaced by an asparagine. Phylogenetic comparisons show that reptilian CBG orthologs share this unexpected property. Glycosylation of this asparagine in zebra finch CBG does not influence its steroid-binding affinity, but we present evidence that it may participate in protein folding and steroid-binding site formation. Substitutions of amino acids within zebra finch CBG that are conserved only in birds reveal how they contribute to their distinct steroid-binding properties, including their high (nanomolar) affinities for glucocorticoids, progesterone, and androgens. As in mammals, a protease secreted by Pseudomonas aeruginosa cleaves CBG in zebra finch plasma within its reactive center loop and disrupts steroid binding, suggesting an evolutionarily conserved property of CBGs. Measurements of CBG mRNA in zebra finch tissues indicate that liver is the main site of plasma CBG production, and anti-zebra finch CBG antibodies cross-react with CBGs in other birds, extending opportunities to study how CBG regulates the actions of glucocorticoids and sex steroids in these species.
Assuntos
Proteínas Aviárias/sangue , Proteínas Aviárias/genética , Aves/sangue , Aves/genética , Evolução Molecular , Transcortina/genética , Transcortina/metabolismo , Adaptação Fisiológica , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Galinhas/sangue , Galinhas/genética , Cristalografia por Raios X , Tentilhões/sangue , Tentilhões/genética , Glicosilação , Modelos Moleculares , Filogenia , Ratos , Homologia de Sequência de Aminoácidos , Pardais/sangue , Pardais/genética , Transcortina/químicaRESUMO
Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.
Assuntos
Ativadores de GTP Fosfo-Hidrolase/metabolismo , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Proteômica/métodos , Plaquetas , Células Cultivadas , Cromatografia de Fase Reversa , Humanos , Marcação por Isótopo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Ativação Plaquetária/fisiologia , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
Dietary carbohydrates raise blood glucose levels, and limiting carbohydrate intake improves glycemia in patients with type 2 diabetes. Low carbohydrate intake (<â¯25 g) allows the body to utilize fat as its primary fuel. As a consequence of increased fatty acid oxidation, the liver produces ketones to serve as an alternative energy source. ß-Hydroxybutyrate (ßHB) is the most abundant ketone. While ßHB has a wide range of functions outside of the pancreas, its direct effects on islet cell function remain understudied. We examined human islet secretory response to acute racemic ßHB treatment and observed increased insulin secretion at a low glucose concentration of 3 mM. Because ßHB is a chiral molecule, existing as both R and S forms, we further studied insulin and glucagon secretion following acute treatment with individual ßHB enantiomers in human and C57BL/6J mouse islets. We found that acute treatment with R-ßHB increased insulin secretion and decreased glucagon secretion at physiological glucose concentrations in both human and mouse islets. Proteomic analysis of human islets treated with R-ßHB over 72 hours showed altered abundance of proteins that may promote islet cell health and survival. Collectively, our data show that physiological concentrations of ßHB influence hormone secretion and signaling within pancreatic islets.
Assuntos
Ácido 3-Hidroxibutírico , Glucagon , Secreção de Insulina , Insulina , Ilhotas Pancreáticas , Camundongos Endogâmicos C57BL , Ácido 3-Hidroxibutírico/farmacologia , Animais , Humanos , Glucagon/metabolismo , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Insulina/metabolismo , Masculino , Glucose/metabolismo , FemininoRESUMO
Population-level variation and mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized. We defined prototypical insulin secretion responses to three macronutrients in islets from 140 cadaveric donors, including those with type 2 diabetes. The majority of donors' islets exhibited the highest insulin response to glucose, moderate response to amino acid, and minimal response to fatty acid. However, 9% of donors' islets had amino acid responses, and 8% had fatty acid responses that were larger than their glucose-stimulated insulin responses. We leveraged this heterogeneity and used multi-omics to identify molecular correlates of nutrient responsiveness, as well as proteins and mRNAs altered in type 2 diabetes. We also examined nutrient-stimulated insulin release from stem cell-derived islets and observed responsiveness to fat but not carbohydrate or protein-potentially a hallmark of immaturity. Understanding the diversity of insulin responses to carbohydrate, protein, and fat lays the groundwork for personalized nutrition.
Assuntos
Diabetes Mellitus Tipo 2 , Secreção de Insulina , Insulina , Ilhotas Pancreáticas , Proteômica , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Masculino , Feminino , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pessoa de Meia-Idade , Nutrientes/metabolismo , Adulto , Glucose/metabolismo , Idoso , Ácidos Graxos/metabolismoRESUMO
Population level variation and molecular mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized despite ramifications for personalized nutrition. Here, we define prototypical insulin secretion dynamics in response to the three macronutrients in islets from 140 cadaveric donors, including those diagnosed with type 2 diabetes. While islets from the majority of donors exhibited the expected relative response magnitudes, with glucose being highest, amino acid moderate, and fatty acid small, 9% of islets stimulated with amino acid and 8% of islets stimulated with fatty acids had larger responses compared with high glucose. We leveraged this insulin response heterogeneity and used transcriptomics and proteomics to identify molecular correlates of specific nutrient responsiveness, as well as those proteins and mRNAs altered in type 2 diabetes. We also examine nutrient-responsiveness in stem cell-derived islet clusters and observe that they have dysregulated fuel sensitivity, which is a hallmark of functionally immature cells. Our study now represents the first comparison of dynamic responses to nutrients and multi-omics analysis in human insulin secreting cells. Responses of different people's islets to carbohydrate, protein, and fat lay the groundwork for personalized nutrition. ONE-SENTENCE SUMMARY: Deep phenotyping and multi-omics reveal individualized nutrient-specific insulin secretion propensity.
RESUMO
Comprehensive molecular and cellular phenotyping of human islets can enable deep mechanistic insights for diabetes research. We established the Human Islet Data Analysis and Sharing (HI-DAS) consortium to advance goals in accessibility, usability, and integration of data from human islets isolated from donors with and without diabetes at the Alberta Diabetes Institute (ADI) IsletCore. Here we introduce HumanIslets.com, an open resource for the research community. This platform, which presently includes data on 547 human islet donors, allows users to access linked datasets describing molecular profiles, islet function and donor phenotypes, and to perform various statistical and functional analyses at the donor, islet and single-cell levels. As an example of the analytic capacity of this resource we show a dissociation between cell culture effects on transcript and protein expression, and an approach to correct for exocrine contamination found in hand-picked islets. Finally, we provide an example workflow and visualization that highlights links between type 2 diabetes status, SERCA3b Ca2+-ATPase levels at the transcript and protein level, insulin secretion and islet cell phenotypes. HumanIslets.com provides a growing and adaptable set of resources and tools to support the metabolism and diabetes research community.
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The choroid plexus (ChP) is a highly vascularized tissue lining the ventricular space of the brain. The ChP generates cerebrospinal fluid (CSF) and forms a protective barrier in the central nervous system (CNS). Recently, a three-dimensional human pluripotent stem cell (hPSC)-derived ChP organoid model has been developed. This model generates cystic structures that are filled with a fluid resembling CSF and are surrounded by an epithelial layer expressing ependymal choroid plexus-specific markers. Here we describe a method to generate these choroid plexus organoids using a commercial kit and methods to extract the CSF-like fluid for use in downstream analysis.
RESUMO
Atmospheric pressure electron capture dissociation (AP-ECD) is an emerging technique with the potential to be a more accessible alternative to conventional ECD/electron transfer dissociation (ETD) methods because it can be implemented using a stand-alone ion source device suitable for use with any existing or future electrospray ionization mass spectrometer. With AP-ECD, no modification of the main instrument is required, so it may easily be retrofitted to instruments not originally equipped with ECD/ETD capabilities. Here, we present our first purpose-built AP-ECD source and demonstrate its use in conjunction with capillary LC for the analysis of substance P, a tryptic digest of bovine serum albumin, and a phosphopeptide mixture. Quality ECD spectra were obtained for all the samples at the low femtomole level, proving that LC-AP-ECD-MS is suitable for the structural analysis of peptides and protein digests, in this case using an unmodified quadrupole time-of-flight mass spectrometer built ca. 2002.
Assuntos
Espectrometria de Massas/instrumentação , Fosfopeptídeos/química , Soroalbumina Bovina/química , Substância P/química , Sequência de Aminoácidos , Animais , Pressão Atmosférica , Bovinos , Cromatografia Líquida/instrumentação , Elétrons , Desenho de Equipamento , Limite de Detecção , Dados de Sequência MolecularRESUMO
Experiments to probe for protein-protein interactions are the focus of functional proteomic studies, thus proteomic data repositories are increasingly likely to contain a large cross-section of such information. Here, we use the Global Proteome Machine database (GPMDB), which is the largest curated and publicly available proteomic data repository derived from tandem mass spectrometry, to develop an in silico protein interaction analysis tool. Using a human histone protein for method development, we positively identified an interaction partner from each histone protein family that forms the histone octameric complex. Moreover, this method, applied to the α subunits of the human proteasome, identified all of the subunits in the 20S core particle. Furthermore, we applied this approach to human integrin αIIb and integrin ß3, a major receptor involved in the activation of platelets. We identified 28 proteins, including a protein network for integrin and platelet activation. In addition, proteins interacting with integrin ß1 obtained using this method were validated by comparing them to those identified in a formaldehyde-supported coimmunoprecipitation experiment, protein-protein interaction databases and the literature. Our results demonstrate that in silico protein interaction analysis is a novel tool for identifying known/candidate protein-protein interactions and proteins with shared functions in a protein network.
Assuntos
Bases de Dados de Proteínas , Mapeamento de Interação de Proteínas/métodos , Proteoma/análise , Proteômica/métodos , Simulação por Computador , Humanos , Proteoma/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
Nitroxyl (HNO) exhibits many important pharmacological effects, including inhibition of platelet aggregation, and the HNO donor Angeli's salt has been proposed as a potential therapeutic agent in the treatment of many diseases including heart failure and alcoholism. Despite this, little is known about the mechanism of action of HNO, and its effects are rarely linked to specific protein targets of HNO or to the actual chemical changes that proteins undergo when in contact with HNO. Here we study the presumed major molecular target of HNO within the body: protein thiols. Cysteine-containing tryptic peptides were reacted with HNO, generating the sulfinamide modification and, to a lesser extent, disulfide linkages with no other long lived intermediates or side products. The sulfinamide modification was subjected to a comprehensive tandem mass spectrometric analysis including MS/MS by CID and electron capture dissociation as well as an MS(3) analysis. These studies revealed a characteristic neutral loss of HS(O)NH2 (65 Da) that is liberated from the modified cysteine upon CID and can be monitored by mass spectrometry. Upon storage, partial conversion of the sulfinamide to sulfinic acid was observed, leading to coinciding neutral losses of 65 and 66 Da (HS(O)OH). Validation of the method was conducted using a targeted study of nitroxylated glyceraldehyde-3-phosphate dehydrogenase extracted from Angeli's salt-treated human platelets. In these ex vivo experiments, the sample preparation process resulted in complete conversion of sulfinamide to sulfinic acid, making this the sole subject of further ex vivo studies. A global proteomics analysis to discover platelet proteins that carry nitroxyl-induced modifications and a mass spectrometric HNO dose-response analysis of the modified proteins were conducted to gain insight into the specificity and selectivity of this modification. These methods identified 10 proteins that are modified dose dependently in response to HNO, whose functions range from metabolism and cytoskeletal rearrangement to signal transduction, providing for the first time a possible mechanistic link between HNO-induced modification and the physiological effects of HNO donors in platelets.
Assuntos
Plaquetas/metabolismo , Proteínas Sanguíneas/análise , Espectrometria de Massas/métodos , Óxidos de Nitrogênio/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Proteínas Sanguíneas/química , Cisteína/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Peptídeos/química , Proteoma/análise , Proteômica , Reprodutibilidade dos Testes , Compostos de Sulfônio/metabolismo , Espectrometria de Massas em TandemRESUMO
Proteomics is the global analysis of proteins in a sample, and its methodologies are commonly applied in life science research. Despite its wide applicability however, proteomics is rarely used as a tool in criminal investigations. Here we present a case where the technique provided key evidence in a case that involved the death of a two-year old girl. The defendant was known to keep exotic snakes, including several venomous species, which led the coroner to probe whether there could be snake venom in the blood of the deceased. One major challenge of the investigation was the overwhelming presence of several blood proteins, such as apolipoprotein and complement proteins, which hinders the detection of less abundant analytes. In a counter-acting strategy, a combination of immunodepletion and fractionation methods was used; the sample was then submitted to tandem mass spectrometry for peptide identification. Using this strategy, 15,000 peptides could be sequenced. However, the subsequent challenge was to differentiate between human and snake proteins, given the genetic similarities that are shared by the two vertebrate species. After a thorough bioinformatics search and manual inspection, we found that<1% of the sequenced peptides could be matched unequivocally to snake proteins, including a well-known venom component, phospholipase A2. This evidence, in part, led to a court-issued search warrant of the defendant's home, followed by his arrest and an eventual guilty plea with formal sentencing to 18 months in prison. The work outlined here is an example of how proteomics technology can help to expand the toolkit for molecular forensics.
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In recent years, the technology and methods widely available for mass spectrometry (MS)-based proteomics have increased in power and potential, allowing the study of protein-level processes occurring in biological systems. Although these methods remain an active area of research, established techniques are already helping answer biological questions. Here, this recent evolution of MS-based proteomics and its applications are reviewed, including standard methods for protein and peptide separation, biochemical fractionation, quantitation, targeted MS approaches such as selected reaction monitoring, data analysis and bioinformatics. Recent research in many of these areas reveals that proteomics has moved beyond simply cataloguing proteins in biological systems and is finally living up to its initial potential - as an essential tool to aid related disciplines, notably health research. From here, there is great potential for MS-based proteomics to move beyond basic research, into clinical research and diagnostics.
Assuntos
Pesquisa Biomédica/tendências , Espectrometria de Massas/tendências , Proteômica/tendências , HumanosRESUMO
An improved in-source atmospheric pressure-electron capture dissociation (AP-ECD) method is described. Building upon the early example of Laprévote's group, photoelectrons generated within a commercial PhotoSpray atmospheric pressure photoionization source are used to induce ECD of multiply charged peptide ions originating from an upstream heated nebulizer device. To attain high sensitivity, the method makes use of a novel electropneumatic-heated nebulizer to assist in the creation and transmission of multiply charged ions from sample solutions. Here, we demonstrate that readily interpretable AP-ECD spectra of infused peptides can be acquired from 100 fmol sample consumed, on a chromatographic time scale, using a conventional quadrupole time-of-flight (Q-ToF) mass spectrometer otherwise incapable of ECD/ETD experiments. Though much work remains to be done to develop and characterize the method, the results indicate that AP-ECD has the potential to be a practical new tool for the mass spectrometric analysis of peptides and proteins.
Assuntos
Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Peptídeos/química , Pressão Atmosférica , Modelos Moleculares , Fotoquímica/métodos , Substância P/químicaRESUMO
Nanomaterials in the blood must mitigate the immune response to have a prolonged vascular residency in vivo. The composition of the protein corona that forms at the nano-biointerface may be directing this, however, the possible correlation of corona composition with blood residency is currently unknown. Here' we report a panel of new soft single molecule polymer nanomaterials (SMPNs) with varying circulation times in mice (t1/2ß ~ 22 to 65 h) and use proteomics to probe protein corona at the nano-biointerface to elucidate the mechanism of blood residency of nanomaterials. The composition of the protein opsonins on SMPNs is qualitatively and quantitatively dynamic with time in circulation. SMPNs that circulate longer are able to clear some of the initial surface-bound common opsonins, including immunoglobulins, complement, and coagulation proteins. This continuous remodelling of protein opsonins may be an important decisive step in directing elimination or residence of soft nanomaterials in vivo.