RESUMEN
The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic-inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase. Compared to a reference stainless-steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra- and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent. By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Monoclonales/química , Cromatografía en Gel , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas/métodosRESUMEN
Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. However, whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unanswered question. Here we report the incorporation of synthetic isotope- and (R)-2'-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. Therefore, we conclude that fdC undergoes C-C bond cleavage in stem cells, leading to the direct re-installation of unmodified dC.
Asunto(s)
Citosina/análogos & derivados , ADN/metabolismo , Desoxicitidina/metabolismo , Animales , Isótopos de Carbono , Línea Celular , Cromatografía Líquida de Alta Presión , Citosina/química , Citosina/metabolismo , ADN/química , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Desmetilación , Desoxicitidina/química , Metilación , Ratones , Isótopos de Nitrógeno , Oxidación-Reducción , Espectrometría de Masas en TándemRESUMEN
Photochromic ligands have been used to control a variety of biological functions, especially in neural systems. Recently, much effort has been invested in the photocontrol of ion channels and G-protein coupled receptors found in the synapse. Herein, we describe the expansion of our photopharmacological approach toward the remote control of an enzyme. Building on hallmark studies dating from the late 1960s, we evaluated photochromic inhibitors of one of the most important enzymes in synaptic transmission, acetylcholinesterase (AChE). Using structure-based design, we synthesized several azobenzene analogues of the well-known AChE inhibitor tacrine (THA) and determined their effects on enzymatic activity. One of our compounds, AzoTHA, is a reversible photochromic blocker of AChE inâ vitro and exâ vivo with high affinity and fast kinetics. As such, AzoTHA can be used to control synaptic transmission on the neuromuscular endplate based on the light-dependent clearance of a neurotransmitter.
Asunto(s)
Acetilcolinesterasa/metabolismo , Tacrina/químicaRESUMEN
The neuronal ceroid lipofuscinoses (NCL) are a group of 13 rare neurodegenerative disorders characterized by accumulation of cellular storage bodies. There are few therapeutic options, and existing tests do not monitor disease progression and treatment response. However, urine biomarkers could address this need. Proteomic analysis of CLN2 patient urine revealed activation of immune response pathways and pathways associated with the unfolded protein response. Analysis of CLN5 and CLN6 sheep model urine showed subtle changes. To confirm and investigate the relevance of candidate biomarkers a targeted LC-MS/MS proteomic assay was created. We applied this assay to additional CLN2 samples as well as other patients with NCL (CLN1, CLN3, CLN5, CLN6, and CLN7) and demonstrated that hexosaminidase-A, aspartate aminotransferase-1, and LAMP1 are increased in NCL samples and betaine-homocysteine S-methyltransferase-1 was specifically increased in patients with CLN2. These proteins could be used to monitor the effectiveness of future therapies aimed at treating systemic NCL disease.
RESUMEN
Classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is caused by a deficiency of tripeptidyl-peptidase-1. In 2017, the first CLN2 enzyme replacement therapy (ERT) cerliponase alfa (Brineura) was approved by the FDA and EMA. The CLN2 disease clinical rating scale (CLN2 CRS) was developed to monitor loss of motor function, language and vision as well as frequency of generalised tonic clonic seizures. Using CLN2 CRS in an open label clinical trial it was shown that Brineura slowed down the progression of CLN2 symptoms. Neurofilament light chain (NfL) is a protein highly expressed in myelinated axons. An increase of cerebrospinal fluid (CSF) and blood NfL is found in a variety of neuroinflammatory, neurodegenerative, traumatic, and cerebrovascular diseases. We analysed CSF NfL in CLN2 patients treated with Brineura to establish whether it can be used as a possible biomarker of response to therapy. Newly diagnosed patients had CSF samples collected and analysed at first treatment dose and up to 12 weeks post-treatment to look at acute changes. Patients on a compassionate use programme who were already receiving ERT for approximately 1yr had CSF samples collected and NfL analysed over the following 1.3 years (2.3 years post-initiation of ERT) to look at long-term changes. All newly diagnosed patients we investigated with classical late infantile phenotype had high NfL levels >2000 pg/ml at start of treatment. No significant change was observed in NfL up to 12 weeks post-treatment. After one year of ERT, two out of six patients still had high NfL levels, but all patients showed a continued decrease, and all had low NfL levels after two years on ERT. NfL levels appear to correspond and predict improved clinical status of patients on ERT and could be useful as a biomarker to monitor neurodegeneration and verify disease modification in CLN2 patients on ERT.
Asunto(s)
Terapia de Reemplazo Enzimático , Lipofuscinosis Ceroideas Neuronales , Biomarcadores , Humanos , Filamentos Intermedios , Lipofuscinosis Ceroideas Neuronales/tratamiento farmacológico , Lipofuscinosis Ceroideas Neuronales/genética , Tripeptidil Peptidasa 1RESUMEN
Tet3 is the main α-ketoglutarate (αKG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess high levels of 5-hydroxymethyl-dC that further increase during neural activity to establish transcriptional plasticity required for learning and memory functions. How αKG, which is mainly generated in mitochondria as an intermediate of the tricarboxylic acid cycle, is made available in the nucleus has remained an unresolved question in the connection between metabolism and epigenetics. We show that in neurons the mitochondrial enzyme glutamate dehydrogenase, which converts glutamate into αKG in an NAD+-dependent manner, is redirected to the nucleus by the αKG-consumer protein Tet3, suggesting on-site production of αKG. Further, glutamate dehydrogenase has a stimulatory effect on Tet3 demethylation activity in neurons, and neuronal activation increases the levels of αKG. Overall, the glutamate dehydrogenase-Tet3 interaction might have a role in epigenetic changes during neural plasticity.
Asunto(s)
Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Dioxigenasas/metabolismo , Glutamato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Neuronas/metabolismo , Animales , Encéfalo/metabolismo , Ciclo del Ácido Cítrico , Dioxigenasas/genética , Epigenómica , Expresión Génica , Glutamato Deshidrogenasa/genética , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Metabolómica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Plasticidad NeuronalRESUMEN
Theories about the origin of life require chemical pathways that allow formation of life's key building blocks under prebiotically plausible conditions. Complex molecules like RNA must have originated from small molecules whose reactivity was guided by physico-chemical processes. RNA is constructed from purine and pyrimidine nucleosides, both of which are required for accurate information transfer, and thus Darwinian evolution. Separate pathways to purines and pyrimidines have been reported, but their concurrent syntheses remain a challenge. We report the synthesis of the pyrimidine nucleosides from small molecules and ribose, driven solely by wet-dry cycles. In the presence of phosphate-containing minerals, 5'-mono- and diphosphates also form selectively in one-pot reactions. The pathway is compatible with purine synthesis, allowing the concurrent formation of all Watson-Crick bases.
Asunto(s)
Nucleósidos de Purina/síntesis química , Nucleósidos de Pirimidina/síntesis química , Ribonucleótidos/síntesis química , Fenómenos Químicos , Hidroxilamina/química , Nucleósidos de Purina/química , Nucleótidos de Purina/síntesis química , Nucleósidos de Pirimidina/química , Nucleótidos de Pirimidina/síntesis química , ARN/síntesis química , Ribosa/químicaRESUMEN
DNA contains not only canonical nucleotides but also a variety of modifications of the bases. In particular, cytosine and adenine are frequently modified. Determination of the exact quantity of these noncanonical bases can contribute to the characterization of the state of a biological system, e.g., determination of disease or developmental processes, and is therefore extremely important. Here, we present a workflow that includes detailed description of critical sample preparation steps and important aspects of mass spectrometry analysis and validation. In this protocol, extraction and digestion of DNA by an optimized spin-column and enzyme-based method are described. Isotopically labeled standards are added in the course of DNA digestion, which allows exact quantification by isotope dilution mass spectrometry. To overcome the major bottleneck of such analyses, we developed a short (~14-min-per-sample) ultra-HPLC (UHPLC) and triple quadrupole mass spectrometric (QQQ-MS) method. Easy calculation of the modification abundance in the genome is possible with the provided evaluation sheets. Compared to alternative methods, the quantification procedure presented here allows rapid, ultrasensitive (low femtomole range) and highly reproducible quantification of different nucleosides in parallel. Including sample preparation and evaluation, quantification of DNA modifications can be achieved in less than a week.
Asunto(s)
Adenina/análisis , Cromatografía Líquida de Alta Presión/métodos , Citosina/análisis , ADN/química , Nucleósidos/análisis , Espectrometría de Masas en Tándem/métodos , Adenina/química , Animales , Línea Celular , Cerebelo/química , Citosina/química , Células HEK293 , Humanos , Hidrólisis , Técnicas de Dilución del Indicador/instrumentación , Marcaje Isotópico/métodos , Ratones , Ratones Endogámicos C57BL , Nucleósidos/química , Células Madre Pluripotentes , Microextracción en Fase Sólida/métodos , Flujo de TrabajoRESUMEN
RNA contains methylated A-base derivatives. A methylation to m6A and then demethylation regulate homeostasis in mRNA. It is assumed that m6A is mainly demethylated by the α-ketoglutarate dependent oxidase ALKBH5. Here we show that ALKBH5 also demethylates the dimethylated adenosine m62A, which is a non-canonical base present in ribosomal RNA.
RESUMEN
Optogenetics and photopharmacology are powerful approaches to investigating biochemical systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is generally a lengthy and cumbersome process. Herein, we describe an in silico screening approach that is designed to propose a small number of optimal combinations. By applying this computational approach to human carbonic anhydrase and a set of three photochromic tethered ligands, the number of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated experimentally. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymatic activity assays, mass spectrometry, and X-ray crystallography. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained.
Asunto(s)
Anhidrasa Carbónica II/química , Optogenética/métodos , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Cisteína/metabolismo , Humanos , Ligandos , Luz , Relación Estructura-ActividadRESUMEN
Investigation of the function of the new epigenetic bases requires the development of stabilized analogues that are stable during base excision repair (BER). Here we report the synthesis of 2'-(R)-fluorinated versions of the phosphoramidites of 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). For oligonucleotides containing 2'-(R)-F-fdC, we show that these compounds cannot be cleaved by the main BER enzyme thymine-DNA glycosylase (TDG).