Optical Control of Cell-Surface and Endomembrane-Exclusive ß-Adrenergic Receptor Signaling.

Beta-adrenergic receptors (ßARs) are G protein-coupled receptors (GPCRs) that mediate catecholamine-induced stress responses, such as heart rate increase and bronchodilation. In addition to signals from the cell surface, ßARs also broadcast non-canonical signaling activities from the cell interior membranes (endomembranes). Dysregulation of these receptor pathways underlies severe pathological conditions. Excessive ßAR stimulation is linked to cardiac hypertrophy, leading to heart failure, while impaired stimulation causes compromised fight or flight stress responses and homeostasis. In addition to plasma membrane ßAR, emerging evidence indicates potential pathological implications of deeper endomembrane ßARs, such as inducing cardiomyocyte hypertrophy and apoptosis, underlying heart failure. However, the lack of approaches to control their signaling in subcellular compartments exclusively has impeded linking endomembrane ßAR signaling with pathology. Informed by the ß1AR-catecholamine interactions, we engineered an efficiently photo-labile, protected hydroxy ß1AR pro-ligand (OptoIso) to trigger ßAR signaling at the cell surface, as well as exclusive endomembrane regions upon blue light stimulation. Not only does OptoIso undergo blue light deprotection in seconds, but it also efficiently enters cells and allows examination of G protein heterotrimer activation exclusively at endomembranes. In addition to its application in the optical interrogation of ßARs in unmodified cells, given its ability to control deep organelle ßAR signaling, OptoIso will be a valuable experimental tool.

Use of N-(4-aminophenyl)piperidine derivatization to improve organic acid detection with supercritical fluid chromatography-mass spectrometry.

The analysis of organic acids in complex mixtures by LC-MS can often prove challenging, especially due to the poor sensitivity of negative ionization mode required for detection of these compounds in their native (i.e., underivatized or untagged) form. These compounds have also been difficult to measure using supercritical fluid chromatography (SFC)-MS, a technique of growing importance for metabolomic analysis, with similar limitations based on negative ionization. In this report, the use of a high proton affinity N-(4-aminophenyl)piperidine derivatization tag is explored for the improvement of organic acid detection by SFC-MS. Four organic acids (lactic, succinic, malic, and citric acids) with varying numbers of carboxylate groups were derivatized with N-(4-aminophenyl)piperidine to achieve detection limits down to 0.5 ppb, with overall improvements in detection limit ranging from 25-to-2100-fold. The effect of the derivatization group on sensitivity, which increased by at least 200-fold for compounds that were detectable in their native form, and mass spectrometric detection are also described. Preliminary investigations into the separation of these derivatized compounds identified multiple stationary phases that could be used for complete separation of all four compounds by SFC. This derivatization technique provides an improved approach for the analysis of organic acids by SFC-MS, especially for those that are undetectable in their native form.

Supercritical Fluid Nanospray Mass Spectrometry: II. Effects on Ionization.

Nanospraying supercritical fluids coupled to a mass spectrometer (nSF-MS) using a 90% supercritical fluid CO2 carrier (sCO2) has shown an enhanced desolvation compared to traditional liquid eluents. Capillaries of 25, 50, and 75 µm internal diameter (i.d.) with pulled emitter tips provided high MS detection sensitivity. Presented here is an evaluation of the effect of proton affinity, hydrophobicity, and nanoemitter tip size on the nSF-MS signal. This was done using a set of primary, secondary, tertiary, and quaternary amines with butyl, hexyl, octyl, and decyl chains as analytes. Each amine class was analyzed individually to evaluate hydrophobicity and proton affinity effects on signal intensity. The system has shown a mass sensitive detection on a linear dynamic range of 0.1-100 µM. Results indicate that hydrophobicity has a larger effect on the signal response than proton affinity. Nanospraying a mixture of all amine classes using the 75 µm emitter has shown a quaternary amine signal not suppressed by competing analytes. Competing ionization was observed for primary, secondary, and tertiary amines. The 75 and 50 µm emitters demonstrated increased signal with increasing hydrophobicity. Surprisingly, the 25 µm i.d. emitter yielded a signal decrease as the alkyl chain length increased, contrary to conventional understanding. Nanospraying the evaporative fluid in a sub-500 nm emitter likely resulted in differences in the ionization mechanism. Results suggest that 90% sCO2 with 9.99% methanol and 0.01% formic acid yielded fast desolvation, high ionization efficiency, and low matrix effect, which could benefit complex biological matrix analysis.

Isobaric 6-plex and tosyl dual tagging for the determination of positional isomers and quantitation of monounsaturated fatty acids using rapid UHPLC-MS/MS.

Isobaric labelling of fatty acids is complicated by chromatographic co-elution of double bond isomers. This produces contaminated spectra which can mask important biological changes. Here two derivatization strategies are combined to improve throughput and produce MS2 reporters which change mass depending on double bond position. A 6-plex isobaric tag is attached to the acid group, followed by the tosylation of the double bond using chloramine-T. These two derivatizations allowed for the chromatographic resolution of nearly all investigated isomers using a 3.5 minute ultrafast method. Further isomer differentiation is achieved upon fragmentation as reporter masses scale with the double bond location. This occurs by a dual-fragmentation route which reveals the isobaric labelling and fragments along the double bond of each analyte. These unique fragments allowed for accurate quantitation of co-isolated double bond isomers where traditional isobaric tags would experience ratio distortion. Saturated and monounsaturated fatty acids were characterized by this rapid 6-plex method and produced an average signal RSD of 9.3% and R2 of 0.99. The method was then used to characterize fatty acid dysregulation upon inhibition of stearoyl CoA desaturase with CAY10566.

Supercritical Fluid Nanospray Mass Spectrometry.

Supercritical fluids are typically electrosprayed using an organic solvent makeup flow to facilitate continuous electrical connection and enhancement of electrospray stability. This results in sample dilution, loss in sensitivity, and potential phase separation. Premixing the supercritical fluid with organic solvent has shown substantial benefits to electrospray efficiency and increased analyte charge state. Presented here is a nanospray mass spectrometry system for supercritical fluids (nSF-MS). This split flow system used small i.d. capillaries, heated interface, inline frit, and submicron emitter tips to electrospray quaternary alkyl amines solvated in supercritical CO2 with a 10% methanol modifier. Analyte signal response was evaluated as a function of total system flow rate (0.5-1.5 mL/min) that is split to nanospray a supercritical fluid with linear flow rates between 0.07 and 0.42 cm/sec and pressure ranges (15-25 MPa). The nSF system showed mass-sensitive detection based on increased signal intensity for increasing capillary i.d. and analyte injection volume. These effects indicate efficient solvent evaporation for the analysis of quaternary amines. Carrier additives generally decreased signal intensity. Comparison of the nSF-MS system to the conventional SF makeup flow ESI showed 10-fold signal intensity enhancement across all the capillary i.d.s. The nSF-MS system likely achieves rapid solvent evaporation of the SF at the emitter point. The developed system combined the benefits of the nanoemitters, sCO2, and the low modifier percentage which gave rise to enhancement in MS detection sensitivity.

Evaluation of nanospray capillary LC-MS performance for metabolomic analysis in complex biological matrices.

LC-MS metabolomic analysis in complex biological matrices may be complicated by degeneracy when using large-bore columns. Degeneracy is the detection of multiple mass spectral peaks from the same analyte due to adduction of salts to the metabolite, dimerization, or loss of neutrals. This introduces interferences to the MS spectra, diminishes quantification, and increases the rate of false identifications. Analysis using 2.1 mm inner diameter (i.d.) columns typically leads to degenerate peaks whereas nanospray using capillary columns (25, 50, and 75 µm i.d.) reduces degeneracy. Optimization of chromatographic parameters of capillary LC for amino acid standards showed the lowest HETP at 1.25 mm/sec across all capillary i.d. columns. Results suggest mass-sensitive detection below the optimum velocity. At faster velocities, concentration-dependent detection occurred across all capillaries. The 2.1 mm i.d. analytical scale column showed the greatest level of degeneracy, particularly in the low signal intensity range. 25 µm i.d. columns showed higher levels of metabolite annotation for the same signal intensity range. It also provided the lowest level of degeneracy, making it best suited for untargeted analysis. The 25 µm i.d. column achieved a peak capacity (nc) of 144 in a 30-minute gradient method with nc decreasing as the column i.d. increased. 75 µm i.d. capillary columns showed the highest signal intensity, which is beneficial for targeted analysis. These effects of chromatographic performance, resolution, and degeneracy profile of capillary and analytical scale columns were compared for metabolomic analyses in complex serum and cell lysate matrices.

Two-dimensional separation techniques using supercritical fluid chromatography.

High-resolution separation systems are essential for the analysis of complex mixtures in a wide variety of application areas. To increase resolution, multidimensional chromatographic techniques have been one key solution. Supercritical fluid chromatography provides a unique opportunity in these multidimensional separations based on its potential for high solvent compatibility, rapid duty cycles, and orthogonality to other separation modes. This review focuses on two-dimensional chromatography methods from the past decade that use supercritical fluid chromatography because of these advantages. Valving schemes and modulation strategies used to interface supercritical fluid chromatography with other liquid chromatography and gas chromatography techniques are described. Particular applications of multidimensional separations using supercritical fluid chromatography for the analysis of oils and chiral separations of pharmaceutical compounds are highlighted. Limitations of and a potential trajectory for supercritical fluid chromatography in this field are also discussed.