Systematic evaluation of benzoylation for liquid chromatography-mass spectrometry analysis of different analyte classes.

LC-MS is an indispensable tool for small molecule analysis in many fields; however, many small molecules require chemical derivatization to improve retention on commonly used reversed-phase columns and increase ionization. Benzoyl chloride (BzCl) derivatization is commonly used for derivatization of primary and secondary amines and phenolic alcohols, though evidence exists that with proper reaction conditions (i.e., specific bases), other hydroxyl groups may be derivatized too. Previous studies have examined BzCl concentration, reaction times, and reaction temperatures for derivatization of amines and phenols for LC-MS analysis; however, use of different bases, base concentration, and extending to conditions to hydroxyl groups for LC-MS analysis has not been well-studied. To address this understudied area and identify reaction conditions for both amino and hydroxyl groups, we performed a systematic study of reaction conditions on multiple classes of potential targets. For selected derivatization methods, detection limits and performance in a variety of biological matrices were assessed. Results highlight the importance of tailoring derivatization methods for a given application as they varied by molecule and/or molecule class. Compared to the standard BzCl method commonly used, alternative methods were identified to better derivatize challenging analytes (glucosamine, choline, cortisol, uridine, cytidine) with detection limits reaching 1100, 9, 38, 170, and 67 nM compared to undetectable, 170, 86, 1000, and 86 nM respectively. Sub-nanomolar detection limits were achieved for norepinephrine with alternative derivatization approaches. Improved derivatization methods for several classes and molecules including nucleosides, steroids, and molecules containing hydroxyl groups were also identified.

Microdialysis coupled with droplet microfluidics and mass spectrometry for determination of neurotransmitters in vivo with high temporal resolution.

Monitoring the concentration fluctuations of neurotransmitters in vivo is valuable for elucidating the chemical signals that underlie brain functions. Microdialysis sampling is a widely used tool for monitoring neurochemicals in vivo. The volume requirements of most techniques that have been coupled to microdialysis, such as HPLC, result in fraction collection times of minutes, thus limiting the temporal resolution possible. Further the time of analysis can become long for cases where many fractions are collected. Previously we have used direct analysis of dialysate by low-flow electrospray ionization-tandem mass spectrometry (ESI-MS/MS) on a triple quadrupole mass spectrometer to monitor acetylcholine, glutamate, and γ-amino-butyric acid to achieve multiplexed in vivo monitoring with temporal resolution of seconds. Here, we have expanded this approach to adenosine, dopamine, and serotonin. The method achieved limits of detection down to 2 nM, enabling basal concentrations of all these compounds, except serotonin, to be measured in vivo. Comparative analysis with LC-MS/MS showed accurate results for all compounds except for glutamate, possibly due to interference for this compound in vivo. Pairing this analysis with droplet microfluidics yields 11 s temporal resolution and can generate dialysate fractions down to 3 nL at rates up to 3 fractions per s from a microdialysis probe. The system is applied to multiplexed monitoring of neurotransmitter dynamics in response to stimulation by 100 mM K+ and amphetamine. These applications demonstrate the suitability of the droplet ESI-MS/MS method for monitoring short-term dynamics of up to six neurotransmitters simultaneously.

Evaluation of Surface Treatments of PDMS Microfluidic Devices for Improving Small-Molecule Recovery with Application to Monitoring Metabolites Secreted from Islets of Langerhans.

Microfluidic devices are becoming an important tool for bioanalysis with applications including studying cell secretion, cell growth, and drug delivery. Small molecules such as drugs, cell products, or nutrients may partition into polydimethylsiloxane (PDMS), a commonly used material for microfluidic devices, potentially leading to poor recovery or inaccurate delivery of such chemicals. To decrease small-molecule partitioning, surface and bulk PDMS treatments have been developed; however, these have been tested on few analytes, or their biocompatibility are unknown. Studies often focus on one analyte, whereas a diversity of chemicals are of interest and possibly affected. In this study, 11 device treatments are tested and applied to 21 biologically relevant small molecules with a variety of chemical structures. Device treatments are characterized using water contact angle measurements and evaluated by measuring recovery of the 21 target analytes using liquid chromatography-mass spectrometry. 1,5-Dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene), a positively charged polymer, produced the least hydrophilic surface and was found to provide the best recovery with most of the analytes having >50% recovery and up to 92% recovery; however, recovery varied by analyte highlighting the importance of analyte diversity rather than targeting a single analyte in evaluating treatments. A polybrene-treated device was applied to investigate secretion from pancreatic islets, which are micro-organs involved in glucose homeostasis and diabetes. Islets secrete small molecules that have been shown to modulate the secretion of islets' main functional products, glucose-regulating hormones. The polybrene treatment enabled the detection of 20 target analytes from islets-on-chip during isosmotic and hypo-osmotic glucose perfusions and resulted in detection of more significant secretion changes compared to untreated PDMS.

Monitoring hormone and small molecule secretion dynamics from islets-on-chip.

Tissue functions such as hormone secretion involve the interplay of multiple chemical signals and metabolic processes over time. Measuring the different components involved is useful in unraveling the interactions, but often requires use of multiple analytical techniques. The challenge of measuring the necessary components with temporal resolution is greater when tissue samples are limited. Here, an accessible microfluidic platform compatible with multiple measurement techniques to monitor cell secretions has been developed. The platform is applied to islets of Langerhans, micro-organs involved in glucose homeostasis and diabetes. The device houses 1 to 8 islets and the perfusion fluid can be controlled to change conditions, e.g., glucose concentration, in seconds. Samples are collected in fractions and split for offline analysis. The device is paired with a scaled-down immunoassay, AlphaLISA, for hormone quantification and liquid chromatography-mass spectrometry for small molecule quantification to study secretion dynamics. The combined system allows the first simultaneous measurement of insulin, glucagon, biogenic amines, and amino acids from islet secretions. The combined measurements revealed correlation in secretion events and differences in timing of release between hormones and biogenic amines and amino acids. These efforts decreased the number of islets required compared to standard approaches, thus decreasing necessary animal use, reagent use, and cost, while increasing information content achievable from one sample. The microfluidic device is a suitable platform for in-depth characterization of secretion from small tissue samples.

Measurement of α-Synuclein Dynamics In Vivo Using Microdialysis with a Novel Homogeneous Immunoassay.

Understanding the regulation of α-synuclein release could be important in better understanding Parkinson's disease development, progression, and treatment. Advances in such studies are hindered by technical challenges that limit the ability to monitor α-synuclein concentration in vivo. We developed a novel α-synuclein microdialysis method coupled with a specific and sensitive immunoassay that requires a small sample volume (1 µL). Using this method, basal α-synuclein level was estimated at 254 ± 78 pM in the striatum of freely moving mice. Additionally, we observed that potassium (75 mM) and nicotine (0.5 mg/kg) administration significantly increased α-synuclein in dialysates. These results provide evidence that the methods we report here can be useful to investigate the physiological roles of α-synuclein and support the idea that α-synuclein is secreted to the extracellular space in a neuronal activity-dependent manner.

Quantitative Characterization of the Neuropeptide Level Changes in Dorsal Horn and Dorsal Root Ganglia Regions of the Murine Itch Models.

Chronic itch can be extremely devastating and, in many cases, difficult to treat. One challenge in treating itch disorders is the limited understanding of the multitude of chemical players involved in the communication of itch sensation from the peripheral to the central nervous system. Neuropeptides are intercellular signaling molecules that are known to be involved in the transmission of itch signals from primary afferent neurons, which detect itch in the skin, to higher-order circuits in the spinal cord and brain. To investigate the role of neuropeptides in transmitting itch signals, we generated two mouse models of chronic itch-Acetone-Ether-Water (AEW, dry skin) and calcipotriol (MC903, atopic dermatitis). For peptide identification and quantitation, we analyzed the peptide content of dorsal root ganglia (DRG) and dorsal horn (DH) tissues from chronically itchy mice using liquid chromatography coupled to tandem mass spectrometry. De novo-assisted database searching facilitated the identification and quantitation of 335 peptides for DH MC903, 318 for DH AEW, 266 for DRG MC903, and 271 for DRG AEW. Of these quantifiable peptides, we detected 30 that were differentially regulated in the tested models, after accounting for multiple testing correction (q ≤ 0.1). These include several peptide candidates derived from neuropeptide precursors, such as proSAAS, protachykinin-1, proenkephalin, and calcitonin gene-related peptide, some of them previously linked to itch. The peptides identified in this study may help elucidate our understanding about these debilitating disorders. Data are available via ProteomeXchange with identifier PXD015949.

Optically Guided Single Cell Mass Spectrometry of Rat Dorsal Root Ganglia to Profile Lipids, Peptides and Proteins.

The mammalian dorsal root ganglia (DRG) are located on the dorsal roots of the spinal nerves and contain cell bodies of primary sensory neurons. DRG cells have been classified into subpopulations based on their size, morphology, intracellular markers, response to stimuli, and neuropeptides. To understand the connections between DRG chemical heterogeneity and cellular function, we performed optically guided, high-throughput single cell profiling using sequential matrix-assisted laser desorption/ionization mass spectrometry (MS) to detect lipids, peptides, and several proteins in individual DRG cells. Statistical analysis of the resulting mass spectra allows stratification of the DRG population according to cellular morphology and, presumably, major cell types. A subpopulation of small cells contained myelin proteins, which are abundant in Schwann cells, and mass spectra of several larger cells contained peaks matching neurofilament, vimentin, myelin basic protein S, and thymosin beta proteins. Of the over 1000 cells analyzed, approximately 78 % produced putative peptide-rich spectra, allowing the population to be classified into three distinct cell types. Two signals with m/z 4404 and 5487 were exclusively observed in a cell type, but could not be matched to results of our previous liquid chromatography-MS analyses.