CSF neurofilament light chain profiling and quantitation in neurological diseases.

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.

Apolipoprotein E O-glycosylation is associated with amyloid plaques and APOE genotype.

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.

Capillary flow-based sample preparation system for metabolomic analysis of mammalian cells in suspension.

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.

Determination of online quenching efficiency for an automated cellular microfluidic metabolomic platform using mass spectrometry based ATP degradation analysis.

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.

Multiple coordination modes of a new ditopic bis(pyrazolyl)methane-based ligand.

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.

Frequency-Modulated Continuous Flow Analysis Electrospray Ionization Mass Spectrometry (FM-CFA-ESI-MS) for Sample Multiplexing.

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.