Observation of Protein Unfolding during pH Ramp Evoked by Lipase-Catalyzed Ester Hydrolysis

Studies of protein folding often involve offline experimental methods such as titrating protein samples with denaturants or equilibrating them in the presence of denaturants. Here, we demonstrate an online analytical approach in which the protein structure is perturbed by a pH ramp evoked by immobilized lipase-catalyzed ester hydrolysis. Changes in the tertiary structure of the protein in response to a pH ramp (from approximately 6.3 to 2.8) are monitored using electrospray ionization mass spectrometry and spectrofluorometry. Interestingly, we discovered a side reaction of ammonium and formate leading to the production of cyanide that occurred during the ionization process. We also found that only certain protein analytes were bound to the formed cyanide species. Nevertheless, this problem was readily overcome by carefully selecting a specific ester substrate. Overall, the alterations in the charge-state distribution and fluorescence intensitycaused by the lipase-induced pH rampreveal conformational transitions in different proteins. In line with previous reports, the acid-induced denaturation of holo-myoglobin occurs through a two-step mechanism, which is supported by identification of protein-unfolding intermediates and the loss of noncovalent protein ligand (heme). The resultsobtained using the developed catalytic methodare also consistent with the results of equilibrium-based experiments, while sample preparation steps are substantially reduced. The proposed approach simplifies the identification of the pH range that has the greatest impact on the protein structure. Thus, it has the potential to be a useful tool for studying protein conformational transitions in the course of pH changes

Flat Disc-Shaped Sampling Probe and Online Re-extraction Apparatus for Mass Spectrometric Analysis of Skin Metabolites: A Proof of Concept

Sweat analysis provides an alternative and noninvasive way of clinical diagnostics. However, sampling and transferring sweat-derived samples to analytical instruments is challenging. In this report, we demonstrate a method utilizing a flat disc-shaped sampling probe, and a compatible re-extraction apparatus coupled online with extractive electrospray ionization (EESI) mass spectrometry (MS). The probe enables sampling of metabolites from a skin area of ∼2.2 cm2. The subsequent online re-extraction and analysis by EESI-MS further mitigates matrix effects caused by sweat components, thus eliminating sample preparation steps. The total analysis time is only 6 min. We have optimized the key parameters of the system, including flow rate of the nebulizing gas in ESI, pressure of the nebulizing gas in pneumatic sample nebulizer, flow rate of the solvent in ESI, and composition of extractant. The standard solutions (0.1 mL) were supplemented with 0.04 M sodium chloride to mimic the matrix effect normally observed in sweat samples. The method has been characterized with four chemical standards (positive-ion mode of histidine, leucine, urocanic acid; negative-ion mode of lactic acid). The limits of detection range from 1.09 to 95.9 nmol. We have further demonstrated the suitability of the method for analysis of sweat. An attempt was made to identify some of the recorded signals by product-ion scan and accurate/exact mass matching

Flat Disc-Shaped Sampling Probe and Online Re-extraction Apparatus for Mass Spectrometric Analysis of Skin Metabolites: A Proof of Concept

Sweat analysis provides an alternative and noninvasive way of clinical diagnostics. However, sampling and transferring sweat-derived samples to analytical instruments is challenging. In this report, we demonstrate a method utilizing a flat disc-shaped sampling probe, and a compatible re-extraction apparatus coupled online with extractive electrospray ionization (EESI) mass spectrometry (MS). The probe enables sampling of metabolites from a skin area of ∼2.2 cm2. The subsequent online re-extraction and analysis by EESI-MS further mitigates matrix effects caused by sweat components, thus eliminating sample preparation steps. The total analysis time is only 6 min. We have optimized the key parameters of the system, including flow rate of the nebulizing gas in ESI, pressure of the nebulizing gas in pneumatic sample nebulizer, flow rate of the solvent in ESI, and composition of extractant. The standard solutions (0.1 mL) were supplemented with 0.04 M sodium chloride to mimic the matrix effect normally observed in sweat samples. The method has been characterized with four chemical standards (positive-ion mode of histidine, leucine, urocanic acid; negative-ion mode of lactic acid). The limits of detection range from 1.09 to 95.9 nmol. We have further demonstrated the suitability of the method for analysis of sweat. An attempt was made to identify some of the recorded signals by product-ion scan and accurate/exact mass matching

Moderate Signal Enhancement in Electrospray Ionization Mass Spectrometry by Focusing Electrospray Plume with a Dielectric Layer around the Mass Spectrometer’s Orifice

Electrospray ionization (ESI) is among the commonly used atmospheric pressure ionization techniques in mass spectrometry (MS). One of the drawbacks of ESI is the formation of divergent plumes composed of polydisperse microdroplets, which lead to low transmission efficiency. Here, we propose a new method to potentially improve the transmission efficiency of ESI, which does not require additional electrical components and complex interface modification. A dielectric plate—made of ceramic—was used in place of a regular metallic sampling cone. Due to the charge accumulation on the dielectric surface, the dielectric layer around the MS orifice distorts the electric field, focusing the charged electrospray cloud towards the MS inlet. The concept was first verified using charge measurement on the dielectric material surface and computational simulation; then, online experiments were carried out to demonstrate the potential of this method in MS applications. In the online experiment, signal enhancements were observed for dielectric plates with different geometries, distances of the electrospray needle axis from the MS inlet, and various compounds. For example, in the case of acetaminophen (15 μM), the signal enhancement was up to 1.82 times (plate B) using the default distance of the electrospray needle axis from the MS inlet (d = 1.5 mm) and 12.18 times (plate C) using a longer distance (d = 7 mm)