RESUMEN
Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed "multiplexed proteome dynamics profiling" (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteoma/análisis , Proteómica/métodos , Azepinas/química , Azepinas/metabolismo , Azepinas/farmacología , Línea Celular , Cromatografía Líquida de Alta Presión , Análisis por Conglomerados , Estradiol/farmacología , Humanos , Marcaje Isotópico , Células Jurkat , Células MCF-7 , Proteínas de Neoplasias/metabolismo , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Proteolisis/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Espectrometría de Masas en Tándem , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.
Asunto(s)
Sistemas CRISPR-Cas/genética , Técnicas de Inactivación de Genes , Proteínas de Ciclo Celular/genética , ADN (Citosina-5-)-Metiltransferasa 1/genética , Exones , Humanos , Mutación , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Factores de Transcripción/genéticaRESUMEN
Multiplexed quantitative proteomics enabled complex workflows to study the mechanisms by which small molecule drugs interact with the proteome such as thermal proteome profiling (TPP) or multiplexed proteome dynamics profiling (mPDP). TPP measures changes in protein thermal stability in response to drug treatment and thus informs on direct targets and downstream regulation events, while the mPDP approach enables the discovery of regulated protein synthesis and degradation events caused by small molecules and other perturbations. The isobaric mass tags available for multiplexed proteomics have thus far limited the efficiency and sensitivity by which such experiments could be performed. Here we evaluate a recent generation of 16-plex isobaric mass tags and demonstrate the sensitive and time efficient identification of Staurosporine targets in HepG2 cell extracts by recording full thermal denaturation/aggregation profiles of vehicle and compound treated samples in a single mass spectrometry experiment. In 2D-TPP experiments, isothermal titration over seven concentrations per temperature enabled comprehensive selectivity profiling of Staurosporine with EC50 values for kinase targets tightly matching to the kinobeads gold standard assay. Finally, we demonstrate time and condition-based multiplexing of dynamic SILAC labeling experiments to delineate proteome-wide effects of the molecular glue Indisulam on synthesis and degradation rates.
Asunto(s)
Preparaciones Farmacéuticas , Proteómica , Espectrometría de Masas , Estabilidad Proteica , ProteomaRESUMEN
Fibrosis can affect any organ, resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by the expansion of connective tissue due to excessive deposition of extracellular matrix (ECM) proteins, including the fibrillar forms of collagen. A significant limitation for discovering cures for fibrosis is the availability of suitable human models and techniques to quantify mature fibrillar collagen deposition as close as possible to human physiological conditions.Here we have extensively characterised an ex vivo cultured human lung tissue-derived, precision-cut lung slices (hPCLS) model using label-free second harmonic generation (SHG) light microscopy to quantify fibrillar collagen deposition and mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint of hPCLS in ex vivo culture.We demonstrate that hPCLS are viable and metabolically active, with mesenchymal, epithelial, endothelial and immune cell types surviving for at least 2â weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed a strong induction of pulmonary fibrosis-related ECM proteins upon transforming growth factor-ß1 (TGF-ß1) stimulation. This upregulation of ECM proteins was not translated into an increased deposition of fibrillar collagen. In support of this observation, we revealed the presence of a pro-ECM degradation activity in our ex vivo cultures of hPCLS, inhibition of which by a metalloproteinase inhibitor resulted in increased collagen deposition in response to TGF-ß1 stimulation.Together the data show that an integrated approach of measuring soluble pro-fibrotic markers alongside quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing anti-fibrotic agents.
Asunto(s)
Microscopía , Fibrosis Pulmonar , Fibrosis , Humanos , Pulmón/patología , Proteómica , Fibrosis Pulmonar/patología , Factor de Crecimiento Transformador beta1RESUMEN
Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96â h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology inâ vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.
Asunto(s)
Antineoplásicos/farmacología , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Proteolisis/efectos de los fármacos , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Benzamidas/química , Benzamidas/farmacocinética , Benzamidas/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Dipéptidos/química , Dipéptidos/farmacocinética , Dipéptidos/farmacología , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Ratones , Estructura Molecular , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Receptores de Estrógenos/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Sitios de Unión , Biocatálisis , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Células MCF-7 , Ratones , Modelos Moleculares , Terapia Molecular Dirigida , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinación , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
The multisubunit cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes, and cancer and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, among their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5(SOCS2)), binds phosphorylated growth hormone receptor as its main substrate. Here, we demonstrate that the components of CRL5(SOCS2) can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated growth hormone receptor peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in Escherichia coli and in Sf21 insect cells, respectively, were used to reconstitute neddylated and unneddylated CRL5(SOCS2) complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike other E3 ligases, CRL5(SOCS2) was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multiangle static light scattering and native MS. Affinities of the protein-protein interactions within the multisubunit complex were measured by isothermal titration calorimetry. A structural model for full-size neddylated and unneddylated CRL5(SOCS2) complexes is supported by traveling wave ion mobility mass spectrometry data.
Asunto(s)
Proteínas Cullin/metabolismo , Conformación Proteica , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Proteínas Cullin/química , Proteínas Cullin/genética , Elonguina , Humanos , Células K562 , Espectrometría de Masas , Modelos Moleculares , Proteína NEDD8 , Unión Proteica , Proteínas Supresoras de la Señalización de Citocinas/química , Proteínas Supresoras de la Señalización de Citocinas/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Ubiquitinas/química , Ubiquitinas/genéticaRESUMEN
A new method for microquantification of phospholipid classes by nanoelectrospray mass spectrometry and stable isotope dilution is presented. The method covers the sum of phosphatidylcholine and sphingomyelin and in addition selectively quantifies phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. A phospholipid class is quantified together with its corresponding lyso-species due to the presence of a common head group. Phospholipids are extracted from tissue lysates, hydrolysed by hydrofluoric acid, and the liberated polar head groups choline, ethanolamine, serine, and inositol are quantified by nanoelectrospray mass spectrometry using deuterium-labeled analogs of the head groups as internal standards. The method is applied to tissue samples of a gastrointestinal tumor and of corresponding non-affected control tissue. In the tumor sample, the abovementioned phospholipids were found at roughly threefold elevated concentrations with a virtually unaltered relative abundance profile.
RESUMEN
Isobaric mass tagging (e.g., TMT and iTRAQ) is a precise and sensitive multiplexed peptide/protein quantification technique in mass spectrometry. However, accurate quantification of complex proteomic samples is impaired by cofragmentation of peptides, leading to systematic underestimation of quantitative ratios. Label-free quantification strategies do not suffer from such an accuracy bias but cannot be multiplexed and are less precise. Here, we compared protein quantification results obtained with these methods for a chemoproteomic competition binding experiment and evaluated the utility of measures of spectrum purity in survey spectra for estimating the impact of cofragmentation on measured TMT-ratios. While applying stringent interference filters enables substantially more accurate TMT quantification, this came at the expense of 30%-60% fewer proteins quantified. We devised an algorithm that corrects experimental TMT ratios on the basis of determined peptide interference levels. The quantification accuracy achieved with this correction was comparable to that obtained with stringent spectrum filters but limited the loss in coverage to <10%. The generic applicability of the fold change correction algorithm was further demonstrated by spiking of chemoproteomics samples into excess amounts of E. coli tryptic digests.
Asunto(s)
Proteínas de Escherichia coli/química , Fragmentos de Péptidos/aislamiento & purificación , Proteómica/normas , Coloración y Etiquetado/normas , Espectrometría de Masas en Tándem/normas , Algoritmos , Escherichia coli/química , Humanos , Células Jurkat , Células K562 , Peso Molecular , Fragmentos de Péptidos/química , Proteómica/métodos , Coloración y Etiquetado/métodosRESUMEN
Recent advances in mass spectrometry-based approaches have enabled the investigation of drug-protein interactions in various ways including the direct detection of drug-target complexes, the examination of drug-induced changes in the target protein structure, and the monitoring of enzymatic target activity. Mass spectrometry-based proteomics methods also permit the unbiased analysis of changes in protein abundance and post-translational modifications induced by drug action. Finally, chemoproteomic affinity enrichment studies enable the deconvolution of drug targets under close to physiological conditions. This review provides an overview of current methods for the characterization of drug-target interactions by mass spectrometry and describes a protocol for chemoproteomic target binding studies using immobilized bioactive molecules.
Asunto(s)
Evaluación Preclínica de Medicamentos , Espectrometría de Masas/métodos , Proteínas/química , Animales , Técnicas de Cultivo de Célula , Cromatografía de Afinidad , Medición de Intercambio de Deuterio , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas , Humanos , Terapia Molecular Dirigida , Unión Proteica , Proteínas/aislamiento & purificación , ProteómicaRESUMEN
Reversible protein phosphorylation is a key mediator for intracellular signal transduction. Here we report an innovative method for accurate, site-specific protein phosphorylation degree determination by nanoLC-ESI-MS/MS. A stable isotope-labeled pair of peptide/phosphopeptide standards with volumetrically defined molar ratio is used as reference, providing an internal standard for both the analyte peptide and the phosphopeptide. For the preparation of one-source peptide/phosphopeptide standards, an aliquot of the labeled phosphopeptide standard is quantitatively dephosphorylated, yielding an equimolar solution of the peptide standard. Subsequently, the two solutions are mixed at a 1:1 or other volumetric ratio, which equals the molar ratio. This procedure assures a defined concentration ratio of both components that is independent from their absolute concentration. We demonstrate the applicability of the one-source peptide/phosphopeptide standard method by determining the phosphorylation degree of the signalling proteins STAT5A/B and STAT6.
Asunto(s)
Enfermedad de Hodgkin/metabolismo , Fosfopéptidos/análisis , Proteómica , Factor de Transcripción STAT5/metabolismo , Factor de Transcripción STAT6/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Cromatografía Liquida , Enfermedad de Hodgkin/patología , Humanos , Fosforilación , Estándares de Referencia , Espectrometría de Masa por Ionización de Electrospray , Células Tumorales CultivadasRESUMEN
Fragmentation of multiple peptides in a single tandem mass scan impairs accuracy of isobaric mass tag based quantification. Consequently, practitioners aim at fragmenting peptide ions with the highest possible purity without compromising on sensitivity and coverage achieved in the experiment. Here we report the first systematic study optimizing delayed fragmentation options on Orbitrap instruments. We demonstrate that by delaying peptide fragmentation to occur closer to the apex of the chromatographic peak in liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments cofragmentation is reduced by 2-fold and peptides are fragmented with 2.8-fold better signal-to-noise ratios. This results in significantly improved accuracy of isobaric mass tag quantification. Further, we measured cofragmentation dependence on isolation width. In comparison to Orbitrap XL instruments the reduced space charging in the Orbitrap Velos enables isolation widths as narrow as 1 Th without impairing coverage, thus substantially reducing cofragmentation. When delayed peptide fragmentation and narrow isolation width settings were both applied, cofragmentation-induced ratio compression could be reduced by 32% on a log2 scale under otherwise identical conditions.
Asunto(s)
Espectrometría de Masas , Péptidos/análisis , Proteínas/metabolismo , Cromatografía Líquida de Alta Presión , Peso Molecular , Relación Señal-RuidoRESUMEN
It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase µLC-ICP-MS or nanoLC-ESI-MS. Using µLC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of >10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga(3+)-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.
Asunto(s)
Cromatografía de Fase Inversa/métodos , Complejos de Coordinación/química , Fragmentos de Péptidos/química , Fosfoproteínas/química , Adsorción , Aluminio/química , Secuencia de Aminoácidos , Ácido Ascórbico/química , Cromatografía de Fase Inversa/instrumentación , Cromatografía de Fase Inversa/normas , Deferoxamina/química , Imidazoles/química , Hierro/química , Datos de Secuencia Molecular , Nanotecnología/métodos , Nanotecnología/normas , Fragmentos de Péptidos/normas , Fosfoproteínas/normas , Fósforo/química , Estándares de Referencia , Titanio/químicaRESUMEN
The current status of de novo sequencing of peptides by MS/MS is reviewed with focus on collision cell MS/MS spectra. The relation between peptide structure and observed fragment ion series is discussed and the exhaustive extraction of sequence information from CID spectra of protonated peptide ions is described. The partial redundancy of the extracted sequence information and a high mass accuracy are recognized as key parameters for dependable de novo sequencing by MS. In addition, the benefits of special techniques enhancing the generation of long uninterrupted fragment ion series for de novo peptide sequencing are highlighted. Among these are terminal (18)O labeling, MS(n) of sodiated peptide ions, N-terminal derivatization, the use of special proteases, and time-delayed fragmentation. The emerging electron transfer dissociation technique and the recent progress of MALDI techniques for intact protein sequencing are covered. Finally, the integration of bioinformatic tools into peptide de novo sequencing is demonstrated.
Asunto(s)
Péptidos/química , Proteómica/métodos , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Biología Computacional , Proteínas/químicaRESUMEN
A novel, widely applicable method for the production of absolutely quantified proteins is described, which can be used as internal standards for quantitative proteomic studies based on mass spectrometry. These standards are recombinant proteins containing an isotope label and selenomethionine. For recombinant protein expression, assembly of expression vectors fitted to cell-free protein synthesis was conducted using the gateway technology which offers fast access to a variety of genes via open reading frame libraries and an easy shuttling of genes between vectors. The proteins are generated by cell-free expression in a medium in which methionine is exchanged against selenomethionine and at least one amino acid is exchanged by a highly stable isotope labeled analogue. After protein synthesis and purification, selenium is used for absolute quantification by element mass spectrometry, while the heavy amino acids in the protein serve as reference in subsequent analyses by LC-ESI-MS or MALDI-MS. Accordingly, these standards are denominated RISQ (for recombinant isotope labeled and selenium quantified) proteins. In this study, a protein was generated containing Lys+6 ([(13)C(6)]-lysine) and Arg+10 ([(13)C(6),(15)N(4)]-arginine) so that each standard tryptic peptide contains a labeled amino acid. Apolipoprotein A1 was synthesized as RISQ protein, and its use as internal standard led to quantification of a reference material within the specified value. Owing to their cell-free expression, RISQ proteins do not contain posttranslational modifications. Thus, correct quantitative data by ESI- or MALDI-MS are restricted to quantifications based on peptides derived from unmodified regions of the analyte protein. Therefore, besides serving as internal standards, RISQ proteins stand out as new tools for quantitative analysis of covalent protein modifications.
Asunto(s)
Proteínas/análisis , Proteínas Recombinantes/análisis , Selenio/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Apolipoproteína A-I/análisis , Apolipoproteína A-I/genética , Escherichia coli/genética , Expresión Génica , Isótopos/análisis , Proteínas/genética , Proteómica/métodos , Proteínas Recombinantes/genética , Selenometionina/análisisRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.
Asunto(s)
Antiinflamatorios/farmacología , Diseño de Fármacos , Inflamación/prevención & control , Leucocitos Mononucleares/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacocinética , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/enzimología , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/enzimología , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Estabilidad de Enzimas , Femenino , Humanos , Inflamación/enzimología , Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Inyecciones Intravenosas , Leucocitos Mononucleares/enzimología , Masculino , Proteolisis , Ratas Sprague-Dawley , Ratas Wistar , Células THP-1 , Técnicas de Cultivo de Tejidos , UbiquitinaciónRESUMEN
An innovative method for the production of absolutely quantified peptide standards is described. These are named phosphorus-based absolutely quantified standard (PASTA) peptides. As the first step, synthetic phosphopeptides are calibrated via a hybrid LC-(ICP+ESI)-MS system. Quantification is achieved by ICP-MS detection of 31P, and identification is performed by ESI-MS. Generation of phosphopeptide standard solutions with this system is demonstrated to provide absolute concentrations with an accuracy better than 10%. The concept was extended to the production of peptide standards by subjecting a PASTA phosphopeptide to gentle and complete dephosphorylation to obtain the cognate PASTA peptide. It is demonstrated that both enzymatic hydrolysis by alkaline or antarctic phosphatase or chemical hydrolysis by hydrofluoric acid can be employed for this purpose. Further, the introduction of one or more stable isotope-labeled amino acids (preferably labeled by 13C, 15N) results in the production of a labeled PASTA peptide, which then can be employed as an internal standard for quantitative analysis by LC-ESI-MS. Using a 1:1 mixture of a stable isotope-labeled PASTA peptide/phosphopeptide pair as dual standard, a quantification between active and inactive recombinant MAP kinase p38alpha was performed by a combination of tryptic digestion and nanoLC-MS.
Asunto(s)
Cromatografía Liquida/métodos , Fosfopéptidos/análisis , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Calibración , Fosfopéptidos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , FosforilaciónRESUMEN
Determination of the protein amount and of the extent of protein phosphorylation is crucial for a variety of research fields, but is not always straightforward. We describe the application of capillary LC-ICP-MS (liquid chromatography-inductively coupled plasma-mass spectrometry) for quantification of phospho-proteins and their phosphorylation degree. Element mass spectrometry is ideally suited for monitor ing and quantification of compounds with heteroelements such as phosphorus and sulphur, particularly because the ICP-MS response is virtually independent from the chemical form of the element. Determination of the phosphorylation stoichiometry, i.e. the relative abundance of the phosphorylated isoforms, can be assessed by the relative abundance of phosphorus compared with sulphur as a marker for the protein amount. Moreover, isotope dilution analysis by post-column addition of a 34S-Spike provides absolute protein quantification with exceptionally high accuracy. Phosphoprotein analysis by capillary LC-ICP-MS may be applied to isolated proteins or protein digests and may include separation of impurities by 1D-SDS-PAGE followed by enzymatic digestion. Alternatively, digestion of complex protein mixtures such as cellular protein extracts allows determination of global, tissue-specific phosphorylation degrees.
Asunto(s)
Espectrometría de Masas/métodos , Fosfoproteínas/análisis , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Animales , Cromatografía Liquida/métodos , Humanos , Microquímica/métodos , FosforilaciónRESUMEN
The serine/threonine protein kinase TBK1 (Tank-binding Kinase-1) is a noncanonical member of the IkB kinase (IKK) family. This kinase regulates signaling pathways in innate immunity, oncogenesis, energy homeostasis, autophagy, and neuroinflammation. Herein, we report the discovery and characterization of a novel potent and highly selective TBK1 inhibitor, GSK8612. In cellular assays, this small molecule inhibited toll-like receptor (TLR)3-induced interferon regulatory factor (IRF)3 phosphorylation in Ramos cells and type I interferon (IFN) secretion in primary human mononuclear cells. In THP1 cells, GSK8612 was able to inhibit secretion of interferon beta (IFNß) in response to dsDNA and cGAMP, the natural ligand for STING. GSK8612 is a TBK1 small molecule inhibitor displaying an excellent selectivity profile and therefore represents an ideal probe to further dissect the biology of TBK1 in models of immunity, neuroinflammation, obesity, or cancer.