RESUMEN
Although the APOE ε4 allele is the strongest genetic risk factor for sporadic Alzheimer's disease (AD), the relationship between apolipoprotein (apoE) and AD pathophysiology is not yet fully understood. Relatively little is known about the apoE protein species, including post-translational modifications, that exist in the human periphery and CNS. To better understand these apoE species, we developed a LC-MS/MS assay that simultaneously quantifies both unmodified and O-glycosylated apoE peptides. The study cohort included 47 older individuals (age 75.6 ± 5.7 years [mean ± standard deviation]), including 23 individuals (49%) with cognitive impairment. Paired plasma and cerebrospinal fluid samples underwent analysis. We quantified O-glycosylation of two apoE protein residues - one in the hinge region and one in the C-terminal region - and found that glycosylation occupancy of the hinge region in the plasma was significantly correlated with plasma total apoE levels, APOE genotype and amyloid status as determined by CSF Aß42/Aß40. A model with plasma glycosylation occupancy, plasma total apoE concentration, and APOE genotype distinguished amyloid status with an AUROC of 0.89. These results suggest that plasma apoE glycosylation levels could be a marker of brain amyloidosis, and that apoE glycosylation may play a role in the pathophysiology of AD.
Asunto(s)
Enfermedad de Alzheimer , Anciano , Anciano de 80 o más Años , Humanos , Péptidos beta-Amiloides/metabolismo , Apolipoproteína E4/líquido cefalorraquídeo , Apolipoproteína E4/genética , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Biomarcadores/metabolismo , Cromatografía Liquida , Genotipo , Glicosilación , Fragmentos de Péptidos/metabolismo , Placa Amiloide , Espectrometría de Masas en TándemRESUMEN
Sample preparation methodology is critical to obtaining reliable data for studying endogenous metabolites. Dependable preparation techniques require separation of cells from culture media, quenching of enzymatic activity, and extraction of metabolites from the cells. Presented here is a simple, rapid, semi-automated metabolomic sample preparation technique for 20 µL samples of RAW 264.7 cells suspended in culture media. This method uses online filter-assisted electroporation-based cell lysis and chilled organic solvent extraction to prepare metabolomic samples from cells in suspension in 2 min. Experiments using an isotopically labeled adenosine triphosphate internal standard were carried out to ensure enzymatic quenching by monitoring the ratio of labeled adenosine diphosphate to adenosine triphosphate. Cells were metabolically labeled with 13C-glucose concurrent with sampling aliquots of the cell suspension over the course of 24 h. Incorporation of 13C into organic acid metabolites such as itaconate Cell lysates was analyzed by nano-reverse-phase liquid chromatography-mass spectrometry (nano-RP-LC-MS), showing incorporation of 13C into organic acid metabolites such as itaconate.
Asunto(s)
Metabolómica/métodos , Animales , Cromatografía Liquida , Glucosa/metabolismo , Espectrometría de Masas , Metaboloma , Ratones , Células RAW 264.7 , Succinatos/metabolismoRESUMEN
As microfluidic cell culture progresses, the need for robust and reproducible intracellular analyses grows. In particular, intracellular metabolites are subject to perturbation and degradation during the lysing process. The reliability of intracellular metabolomic analysis in microfluidic devices depends on the preservation of metabolite integrity during sample preparation and storage. Described here is a novel automated microfluidic system exhibiting the necessary rapid cellular lysis and quenching of enzymatic activity. Quenching efficiency was assessed using a novel ratiometric MALDI-MS-based assay of exogenous isotopic adenosine triphosphate (ATP) hydrolysis to isotopic adenosine diphosphate (ADP) as a marker of metabolite degradation. The lysis system of the microfluidic device was enhanced using a Peltier cooler to chill the lysate and quench aberrant enzymatic activity. Parameter optimization (flow rate, collection time, and temperature control) improved the endogenous and exogenous ADP/ATP ratios by 44.9% and 39.8% respectively consistent with traditional quenching techniques. The effects of chilling/quenching on metabolism were evaluated resulting in over 500 significant features compared to non-chilled from untargeted capillary LC-MS metabolomic analyses. These include increased levels of tryptophan, histidine, and pyruvate as well as decreased levels in UDP-N-acetylglucosamine. The results illustrate the need for both rapid lysis and quenching in microfluidic cell culture platforms. Graphical abstract.
Asunto(s)
Adenosina Trifosfato/metabolismo , Metabolómica , Microfluídica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adenosina Difosfato/metabolismo , AutomatizaciónRESUMEN
A method for multiplexed sample analysis by mass spectrometry without the need for chemical tagging is presented. In this new method, each sample is pulsed at unique frequencies, mixed, and delivered to the mass spectrometer while maintaining a constant total flow rate. Reconstructed ion currents are then a time-dependent signal consisting of the sum of the ion currents from the various samples. Spectral deconvolution of each reconstructed ion current reveals the identity of each sample, encoded by its unique frequency, and its concentration encoded by the peak height in the frequency domain. This technique is different from other approaches that have been described, which have used modulation techniques to increase the signal-to-noise ratio of a single sample. As proof of concept of this new method, two samples containing up to 9 analytes were multiplexed. The linear dynamic range of the calibration curve was increased with extended acquisition times of the experiment and longer oscillation periods of the samples. Because of the combination of the samples, salt had little effect on the ability of this method to achieve relative quantitation. Continued development of this method is expected to allow for increased numbers of samples that can be multiplexed.
Asunto(s)
Indicadores y Reactivos/química , Espectrometría de Masas/métodos , Biología de Sistemas/métodos , Animales , Glicómica/métodos , Humanos , Marcaje Isotópico/métodos , Lipidómica/métodos , Lípidos/análisis , Compuestos Orgánicos/análisis , Polisacáridos/análisis , Proteínas/análisis , Proteómica/métodosRESUMEN
Although neurofilament light chain is a well-known marker of neuronal damage, its characterization at the proteoform level is underdeveloped. Here, we describe a new method to profile and quantify neurofilament light chain in plasma at the peptide level, using three in-house monoclonal antibodies targeting distinct protein domains and nano-liquid chromatography coupled to high-resolution tandem mass spectrometry. This study profiled and compared plasma neurofilament light chain to CSF in 102 older individuals (73.9 ± 6.3 years old), 37 of which had a clinical dementia rating greater than 0. We observed elevated neurofilament light chain in preclinical Alzheimer's disease plasma for two measures (NfL101 and NfL324) and CSF for seven measures (NfL92, NfL101, NfL117, NfL137, NfL148, NfL165 and NfL530). We found five plasma peptides (NfL92, NfL101, NfL117, NfL324 and NfL530) significantly associated with age and two (NfL148 and NfL324) with body mass index.
RESUMEN
Neurofilament light chain is an established marker of neuroaxonal injury that is elevated in CSF and blood across various neurological diseases. It is increasingly used in clinical practice to aid diagnosis and monitor progression and as an outcome measure to assess safety and efficacy of disease-modifying therapies across the clinical translational neuroscience field. Quantitative methods for neurofilament light chain in human biofluids have relied on immunoassays, which have limited capacity to describe the structure of the protein in CSF and how this might vary in different neurodegenerative diseases. In this study, we characterized and quantified neurofilament light chain species in CSF across neurodegenerative and neuroinflammatory diseases and healthy controls using targeted mass spectrometry. We show that the quantitative immunoprecipitation-tandem mass spectrometry method developed in this study strongly correlates to single-molecule array measurements in CSF across the broad spectrum of neurodegenerative diseases and was replicable across mass spectrometry methods and centres. In summary, we have created an accurate and cost-effective assay for measuring a key biomarker in translational neuroscience research and clinical practice, which can be easily multiplexed and translated into clinical laboratories for the screening and monitoring of neurodegenerative disease or acute brain injury.
RESUMEN
A new ditopic ligand, N-(2,2-bis(pyrazolyl)ethyl)-2,2-bis(pyrazolyl)acetamide ((pz)2CH-C(O)-NH-CH2-CH(pz)2, L4Pz, pz = pyrazolyl ring), comprising two bis(pyrazolyl)methane donor groups linked via an amide bridge, has been prepared from the reaction of HOOCCH(pz)2 and H2NCH2CH(pz)2. The ligand coordinates to various metallic salts (i.e. AgO3SCF3, PdCl2, Re(CO)5Br, and Fe(BF4)2), in either a κ2-µ-κ2 or a κ3-µ-κ2 fashion, depending on the coordination preferences of the metallic center. These compounds were characterized by NMR, UV-Vis and IR spectroscopy, and in solid state by single crystal X-ray diffraction. In the case of silver(i), a mono-dimensional coordination polymer was obtained, while the others were found to be discrete complexes. The synthesis and characterization of a heterobimetallic complex is also described. In solid state, all compounds are associated into supramolecular architectures via hydrogen bonding and pyrazolyl embrace interactions.