RESUMO
Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds.
Assuntos
Movimento Celular , Queratinócitos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cicatrização , Aminoácidos/metabolismo , Animais , Linhagem Celular Transformada , Adesões Focais/genética , Adesões Focais/metabolismo , Humanos , Queratinócitos/patologia , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Pseudópodes/genética , Pseudópodes/metabolismo , Pele/enzimologia , Pele/lesões , Pele/patologiaRESUMO
Immunogenicity of biomolecules is one of the largest concerns in biological therapeutic drug development. Adverse immune responses as a result of immunogenicity to biotherapeutics range from mild hypersensitivity reactions to potentially life-threatening anaphylactic reactions and can negatively impact human health and drug efficacy. Numerous confounding patient-, product- or treatment-related factors can influence the development of an immune reaction against therapeutic proteins. The goal of this study was to investigate the relationship between pre-existing drug reactivity (PE-ADA), individual immunogenetics (MHC class II haplotypes), and development of treatment-induced antidrug antibodies (TE-ADA) in cynomolgus macaque. PE-ADA refers to the presence of antibodies immunoreactive against the biotherapeutic in treatment-naïve individuals. We observed that PE-ADA frequency against four different bispecific antibodies in naïve cynomolgus macaque is similar to that reported in humans. Additionally, we report a trend towards an increased incidence of TE-ADA development in macaques with high PE-ADA levels. In order to explore the relationship between MHC class II alleles and risk of ADA development, we obtained full-length MHC class II sequences from 60 cynomolgus macaques in our colony. We identified a total of 248 DR, DP, and DQ alleles and 236 unique haplotypes in our cohort indicating a genetically complex set of animals potentially reflective of the human population. Based on our observations, we propose the evaluation of the magnitude/frequency of pre-existing reactivity and consideration of MHC class II genetics as additional useful tools to understand the immunogenic potential of biotherapeutics.
Assuntos
Anticorpos Biespecíficos/imunologia , Hipersensibilidade a Drogas/imunologia , Genes MHC da Classe II/genética , Genes MHC da Classe II/imunologia , Imunogenética , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/sangue , Hipersensibilidade a Drogas/genética , Frequência do Gene , Haplótipos , Macaca fascicularis , Masculino , FilogeniaRESUMO
Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a secreted protein which regulates serum LDL cholesterol. It circulates in human and rodent serum in an intact form and a major truncated form. Previous in vitro studies involving the expression of human PCSK9 genetic variants and in vivo studies of furin knockout mice suggest that the truncated form is a furin cleavage product. However, the circulating truncated form of PCSK9 has not been isolated and characterized. Utilizing antibodies which bind to either the catalytic domain or the C-terminal domain of PCSK9, the truncated PCSK9 was isolated from serum. MS was used to determine that this form of PCSK9 is a product of in vivo cleavage at Arg218 resulting in pyroglutamic acid formation of the nascent N terminus corresponding to Gln219 of intact PCSK9. We also determined that the truncated PCSK9 in serum lacked the N-terminal segment which contains amino acids critical for LDL receptor binding. A truncated PCSK9, expressed and purified from HEK293 cells with identical composition as the circulating truncated protein, was not active in inhibition of LDL uptake by HepG2 cells. These studies provide a definitive characterization of the composition and activity of the truncated form of PCSK9 found in human serum.
Assuntos
Pró-Proteína Convertase 9 , Animais , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Camundongos Knockout , Pró-Proteína Convertase 9/sangue , Pró-Proteína Convertase 9/química , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/isolamento & purificação , Domínios ProteicosRESUMO
Capillary zone electrophoresis (CZE) with an electrokinetically pumped sheath-flow nanospray interface was coupled with a high-resolution Q-Exactive mass spectrometer for the analysis of culture filtrates from Mycobacterium marinum. We confidently identified 22 gene products from the wildtype M. marinum secretome in a single CZE-tandem mass spectrometry (MS/MS) run. A total of 58 proteoforms were observed with post-translational modifications including signal peptide removal, N-terminal methionine excision, and acetylation. The conductivities of aqueous acetic acid and formic acid solutions were measured from 0.1% to 100% concentration (v/v). Acetic acid (70%) provided lower conductivity than 0.25% formic acid and was evaluated as low ionic-strength and a CZE-MS compatible sample buffer with good protein solubility.
Assuntos
Proteínas de Bactérias/química , Mycobacterium marinum/química , Eletroforese Capilar , Peptídeos/química , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Cerebellar motor coordination and cerebellar Purkinje cell synaptic function require metabotropic glutamate receptor 1 (mGluR1, Grm1). We used an unbiased proteomic approach to identify protein partners for mGluR1 in cerebellum and discovered glutamate receptor δ2 (GluRδ2, Grid2, GluΔ2) and protein kinase Cγ (PKCγ) as major interactors. We also found canonical transient receptor potential 3 (TRPC3), which is also needed for mGluR1-dependent slow EPSCs and motor coordination and associates with mGluR1, GluRδ2, and PKCγ. Mutation of GluRδ2 changes subcellular fractionation of mGluR1 and TRPC3 to increase their surface expression. Fitting with this, mGluR1-evoked inward currents are increased in GluRδ2 mutant mice. Moreover, loss of GluRδ2 disrupts the time course of mGluR1-dependent synaptic transmission at parallel fiber-Purkinje cells synapses. Thus, GluRδ2 is part of the mGluR1 signaling complex needed for cerebellar synaptic function and motor coordination, explaining the shared cerebellar motor phenotype that manifests in mutants of the mGluR1 and GluRδ2 signaling pathways.
Assuntos
Neurônios/fisiologia , Proteína Quinase C/fisiologia , Células de Purkinje/fisiologia , Receptores de Glutamato/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Transmissão Sináptica/fisiologia , Canais de Cátion TRPC/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Mutação/fisiologia , Técnicas de Patch-Clamp , Fenótipo , Receptores de Superfície Celular/fisiologia , Receptores de Glutamato/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Solubilidade , Frações Subcelulares/metabolismo , Frações Subcelulares/fisiologiaRESUMO
Capillary zone electrophoresis (CZE)-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was applied for rapid top-down intact protein characterization. A mixture containing four model proteins (cytochrome c, myoglobin, bovine serum albumin (BSA), and ß-casein) was used as the sample. The CZE-ESI-MS system was first evaluated with the mixture. The four model proteins and five impurities were baseline-separated within 12 min. The limits of detection [signal-to-noise ratio (S/N) = 3] of the four model proteins ranged from 20 (cytochrome c) to 800 amol (BSA). The relative standard deviations of migration time and intensity for the four model proteins were less than 3% and 30%, respectively, in quintuplicate runs. CZE-ESI-MS/MS was then applied for top-down characterization of the mixture. Three of the model proteins (all except BSA) and an impurity (bovine transthyretin) were confidently identified by database searching of the acquired tandem spectra from protein fragmentation. Modifications including phosphorylation, N-terminal acetylation, and heme group binding were identified.
Assuntos
Caseínas/análise , Citocromos c/análise , Miosinas/análise , Soroalbumina Bovina/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Caseínas/química , Bovinos , Citocromos c/química , Eletroforese Capilar/métodos , Cavalos , Miosinas/química , Soroalbumina Bovina/química , Fatores de TempoRESUMO
There is strong experimental evidence associating cathepsin S with the pathogenesis of atherosclerosis, with emerging data to support its role in diseases such as abdominal aortic aneurysm, obesity, and type 2 diabetes. To further our understanding of cathepsin S, we have developed a novel sandwich immunoassay to measure the mature form of cathepsin S in plasma (mean values from 12 healthy donors of 53±17ng/ml, range=39-102). We also developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure in vitro cathepsin S activity to compare activity levels with the protein mass levels determined by enzyme-linked immunosorbent assay (ELISA). Interestingly, we observed that only 0.4 to 1.1% of circulating cathepsin S was enzymatically active. We subsequently demonstrated that the attenuated activity we observed resulted from binding between cathepsin S and its endogenous inhibitor cystatin C in plasma. These data were obtained through immunoprecipitation coupled with either Western blotting analysis or in-gel tryptic digestion and LC-MS/MS characterization of Coomassie-stained gel bands. Although many laboratories have explored the relationship between cathepsin S and cystatin C, this is the first study to demonstrate their association in human circulation, a finding that could prove to be important in furthering our understanding of cathepsin S biology.
Assuntos
Catepsinas/sangue , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Espectrometria de Massas em Tandem , Anticorpos Monoclonais/imunologia , Western Blotting , Catepsinas/genética , Catepsinas/metabolismo , Cistatina C/genética , Cistatina C/metabolismo , Humanos , Imunoprecipitação , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The peptide hormone ghrelin is the only known protein modified with an O-linked octanoyl side group, which occurs on its third serine residue. This modification is crucial for ghrelin's physiological effects including regulation of feeding, adiposity, and insulin secretion. Despite the crucial role for octanoylation in the physiology of ghrelin, the lipid transferase that mediates this novel modification has remained unknown. Here we report the identification and characterization of human GOAT, the ghrelin O-acyl transferase. GOAT is a conserved orphan membrane-bound O-acyl transferase (MBOAT) that specifically octanoylates serine-3 of the ghrelin peptide. Transcripts for both GOAT and ghrelin occur predominantly in stomach and pancreas. GOAT is conserved across vertebrates, and genetic disruption of the GOAT gene in mice leads to complete absence of acylated ghrelin in circulation. The occurrence of ghrelin and GOAT in stomach and pancreas tissues demonstrates the relevance of GOAT in the acylation of ghrelin and further implicates acylated ghrelin in pancreatic function.
Assuntos
Aciltransferases/metabolismo , Grelina/metabolismo , Acilação , Aciltransferases/genética , Animais , Caprilatos/metabolismo , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Sequência Conservada , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Grelina/sangue , Grelina/genética , Humanos , Dados de Sequência Molecular , Pâncreas/enzimologia , Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina/metabolismo , Estômago/enzimologiaRESUMO
Precise elucidation of the antigen sequences for T cell immunosurveillance greatly enhances our ability to understand and modulate humoral responses to viral infection or active immunization. Mass spectrometry is used to identify 526 unique sequences from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein extracellular domain in a complex with human leukocyte antigen class II molecules on antigen-presenting cells from a panel of healthy donors selected to represent a majority of allele usage from this highly polymorphic molecule. The identified sequences span the entire spike protein, and several sequences are isolated from a majority of the sampled donors, indicating promiscuous binding. Importantly, many peptides derived from the receptor binding domain used for cell entry are identified. This work represents a precise and comprehensive immunopeptidomic investigation with the SARS-CoV-2 spike glycoprotein and allows detailed analysis of features that may aid vaccine development to end the current coronavirus disease 2019 (COVID-19) pandemic.
Assuntos
Epitopos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Células Cultivadas , Células Dendríticas/imunologia , Epitopos/química , Feminino , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/imunologia , Polimorfismo Genético , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro. All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.
Assuntos
Anticorpos Monoclonais/imunologia , Formação de Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Anticorpos Monoclonais/farmacologia , Formação de Anticorpos/efeitos dos fármacos , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacosRESUMO
Fibroblast growth factor-21 (FGF-21) is a metabolic regulator that can influence glucose and lipid control in diabetic rodents and primates. We demonstrate that betaKlotho is an integral part of an activated FGF-21-betaKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor. Cells lacking betaKlotho did not respond to FGF-21; the introduction of betaKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells. Interestingly, FGF-21-mediated effects are heparin independent suggesting that betaKlotho plays a role in FGF-21 activity similar to the one played by heparin in the signaling of conventional FGFs. Moreover, in addition to conferring specificity for FGF-21, betaKlotho appears to support FGF-19 activity and mediates the receptor selectivity profile of FGF-19. All together, these results indicate that betaKlotho and FGFRs form the cognate FGF-21 receptor complex, mediating FGF-21 cellular specificity and physiological effects.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Células 3T3-L1 , Animais , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Proteínas Klotho , Camundongos , Ligação ProteicaRESUMO
Cerebrospinal fluid (CSF) provides an important source of potential biomarkers for brain disorders and therapeutic drug development. Applications of proteomic technology to the identification and quantification of proteins in CSF are increasing rapidly. Key to obtaining reproducible and reliable data about protein levels in CSF are standardization of methods for sample collection, storage, and subsequent sample processing. Methods are described here for all steps of sample processing for a number of different proteomic approaches.
Assuntos
Líquido Cefalorraquidiano/química , Proteínas/isolamento & purificação , Proteômica , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Humanos , Proteínas/análise , Reprodutibilidade dos Testes , Manejo de Espécimes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Pharmaceutical companies and regulatory agencies are pursuing biomarkers as a means to increase the productivity of drug development. Quantifying differential levels of proteins from complex biological samples like plasma or cerebrospinal fluid is one specific approach being used to identify markers of drug action, efficacy, toxicity, etc. Academic investigators are also interested in markers that are diagnostic or prognostic of disease states. We report a comprehensive, fully automated, and label-free approach to relative protein quantification including: sample preparation, proteolytic protein digestion, LCMS/MS data acquisition, de-noising, mass and charge state estimation, chromatographic alignment, and peptide quantification via integration of extracted ion chromatograms. Additionally, we describe methods for transformation and normalization of the quantitative peptide levels in multiplexed measurements to improve precision for statistical analysis. Lastly, we outline how the described methods can be used to design and power biomarker discovery studies.
Assuntos
Biomarcadores/análise , Cromatografia Líquida de Alta Pressão/métodos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Interpretação Estatística de Dados , Humanos , Proteoma/análise , Proteômica/estatística & dados numéricosRESUMO
Streptozotocin (STZ) inhibits O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from proteins. The active site of the enzyme was recently proposed to include aspartates 174, 175, and 177, with STZ inhibition via a transition state analog. We explored the effect of STZ on the tryptic peptide digest pattern of O-GlcNAcase. LC/MS/MS analysis demonstrated that STZ modified two areas of the enzyme. One peptide, LGCFEIAK (894-901), in a C-terminal region previously proposed to possess O-GlcNAcase activity, was methylated by STZ. Another peptide, EYEIEFIYIASPGLDITFSNPK (128-149), was detected only after treatment with STZ and was in an N-terminal region, overlapping a glutamate-rich area containing an adjacent phenylalanine residue. No covalent modification of this peptide could be demonstrated. Detection of this peptide after treatment with STZ was accompanied by the simultaneous inability to detect the nearby peptide KLDQVSQFGCR (157-167), which contains a cysteine residue recently shown to be essential for enzymatic activity. To determine which of the first two peptides might also be important for O-GlcNAcase activity, site-specific mutagenesis was performed. Mutation of the N-terminal phenylalanine and serine residues resulted in almost complete inhibition of activity. In contrast, mutation of conserved C-terminal glycine and cysteine residues caused little inhibition of enzymatic activity. Together, these data extend the region of the active site N-terminally and give independent evidence to support the idea that STZ inhibits O-GlcNAcase through formation of a transition state analog that resides in the active site of the enzyme and in doing so alters its conformation and ensuing tryptic digest pattern.
Assuntos
Acetilglucosaminidase/metabolismo , Histona Acetiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , Peptídeos/efeitos dos fármacos , Estreptozocina/farmacologia , Acetilglucosaminidase/química , Sequência de Aminoácidos , Animais , Diabetes Mellitus Experimental , Histona Acetiltransferases/química , Humanos , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Especificidade por Substrato , beta-N-Acetil-HexosaminidasesRESUMO
Capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) was used for analysis of reduced antibodies. We first developed a simple protocol to condition commercial linear-polyacrylamide coated capillaries for use in top-down proteomics. We then suspended reduced antibodies in a solution of 35% acetic acid, 50% acetonitrile in water. Heavy and light chains were baseline resolved within 10 min and with 3-30 µg/mL detection limits using a 0.1% aqueous formic acid background electrolyte. Quintuplicate runs of a two-antibody mixture produced relative standard deviations of â¼1% in migration time and 10% in peak amplitudes. Resolution was further improved for the two-antibody mixture by using 5% acetic acid as the background electrolyte, highlighting the potential of capillary electrophoresis-mass spectrometry for analysis of antibody mixtures.
Assuntos
Anticorpos Monoclonais/análise , Espectrometria de Massas em Tandem/métodos , Eletroforese Capilar/métodos , Fatores de TempoRESUMO
A method is described for the quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Known limitations imposed by crystal heterogeneity, peptide ionization differences, data handling, and protein quantification with MALDI-TOF mass spectrometry are addressed in this method with a "seed crystal" protocol for analyte-matrix formation, the use of internal protein standards, and a software package called maldi_quant. The seed crystal protocol, a new variation of the fast-evaporation method, minimizes crystal heterogeneity and allows for consistent collection of protein spectra. The software maldi_quant permits rapid and automated analysis of peak intensity data, normalization of peak intensities to internal standards, and peak intensity deconvolution and estimation for vicinal peaks. Using insulin proteins in a background of other unrelated peptides, this method shows an overall coefficient of variance of 4.4%, and a quantitative working range of 0.58-37.5 ng bovine insulin per spot. Coupling of this methodology to powerful analytical procedures such as immunoprecipitation is likely to lead to the rapid and reliable quantification of biologically relevant proteins and their closely related variants.
Assuntos
Peptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bovinos , Insulina/análise , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normasRESUMO
De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.
Assuntos
Anticorpos Monoclonais/química , Isoleucina/química , Leucina/química , Análise de Sequência de Proteína/métodos , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Quimotripsina/metabolismo , Isoleucina/análise , Leucina/análise , Leucil Aminopeptidase/metabolismo , Dados de Sequência Molecular , Agregados ProteicosRESUMO
A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).
Assuntos
Biomarcadores/metabolismo , Encéfalo/metabolismo , Doença de Parkinson/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , alfa-Sinucleína/análise , alfa-Sinucleína/metabolismo , Idoso de 80 Anos ou mais , Western Blotting , Cadáver , Estudos de Casos e Controles , Cromatografia Líquida , Interpretação Estatística de Dados , Feminino , HumanosRESUMO
Recent improvements in the mass accuracy and resolution of mass spectrometers have led to renewed interest in label-free quantification using data from the primary mass spectrum (MS1) acquired from data-dependent proteomics experiments. The capacity for higher specificity quantification of peptides from samples enriched for proteins of biological interest offers distinct advantages for hypothesis generating experiments relative to immunoassay detection methods or prespecified peptide ions measured by multiple reaction monitoring (MRM) approaches. Here we describe an evaluation of different methods to post-process peptide level quantification information to support protein level inference. We characterize the methods by examining their ability to recover a known dilution of a standard protein in background matrices of varying complexity. Additionally, the MS1 quantification results are compared to a standard, targeted, MRM approach on the same samples under equivalent instrument conditions. We show the existence of multiple peptides with MS1 quantification sensitivity similar to the best MRM peptides for each of the background matrices studied. Based on these results we provide recommendations on preferred approaches to leveraging quantitative measurements of multiple peptides to improve protein level inference.
RESUMO
The hormone ghrelin is a unique signaling peptide with powerful metabolic effects, mediated by its acylated forms. The acyl modification of ghrelin is unique in that it takes place via a susceptible ester linkage in the conserved serine-3 of ghrelin and is composed principally of octanoyl and, to lesser extent, decanoyl fatty acids. The nature of this ester linkage makes it susceptible to esterases, which convert it to its des-acyl forms, and, if not adequately inhibited, the conversion to des-acyl ghrelin, particularly post sample collection, can lead to artifactual and misleading results. Here, we describe sample processing and mass spectrometric methodologies for the accurate and simultaneous quantification of acylated and des-acylated forms of ghrelin. We exploited these methodologies (1) to characterize circulating and tissue-specific forms of acyl and des-acyl ghrelin, (2) to optimize a cell system for acyl ghrelin production and search for the enzyme responsible for ghrelin's acylation, and (3) to demonstrate that GOAT is ghrelin's O-acyl transferase.