Using Metalloporphyrin Nanosensors for In Situ Monitoring and Measurement of Nitric Oxide and Peroxynitrite in a Single Human Neural Progenitor Cell.

Nitric oxide (NO) is an inorganic signaling molecule that plays a crucial role in the regulation of numerous physiological functions. An oxidation product of the cytoprotective NO is cytotoxic peroxynitrite (ONOO-). In biological systems, the concentrations of NO and ONOO- are typically transient, ranging from nanomolar to micromolar, and these increases are normally followed by a swift return to their basal levels due to their short life spans. To understand the vital physiological role of NO and ONOO- in vitro and in vivo, sensitive and selective methods are necessary for direct and continuous NO and ONOO- measurements in real time. Because electrochemical methods can be adjusted for selectivity, sensitivity, and biocompatibility in demanding biological environments, they are suitable for real-time monitoring of NO and ONOO- release. Metalloporphyrin nanosensors, described here, have been designed to measure the concentration of NO and ONOO- produced by a single human neural progenitor cell (hNPC) in real time. These nanosensors (200-300 nm in diameter) can be positioned accurately in the proximity of 4-5 ± 1 µm from an hNPC membrane. The response time of the sensors is better than a millisecond, while detection limits for NO and ONOO- are 1 × 10-9 and 3 × 10-9 mol/L, respectively, with a linear concentration response of up to about 1 µM. The application of these metalloporphyrin nanosensors for the efficient measurement of the concentrations of NO and ONOO- in hNPCs is demonstrated, providing an opportunity to observe in real time the molecular changes of the two signaling molecules in situ.

Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry.

Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., ß-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.

Absolute Quantitation of Proteins by Coulometric Mass Spectrometry.

Accurate quantification is essential in the fields of proteomics, clinical assay, and biomarker discovery. Popular methods for absolute protein quantitation by mass spectrometry (MS) involve the digestion of target protein and employ isotope-labeled peptide internal standards to quantify chosen surrogate peptides. Although these methods have gained success, syntheses of isotope-labeled peptides are time-consuming and costly. To eliminate the need for using standards or calibration curves, herein we present a coulometric mass spectrometric (CMS) approach for absolute protein quantitation, based on the electrochemical oxidation of a surrogate peptide combined with mass spectrometric measurement of the oxidation yield. To demonstrate the utility of this method, several proteins were analyzed such as model proteins ß-casein, and apomyoglobin as well as circadian clock protein KaiB isolated from Escherichia coli. In our experiment, tyrosine-containing peptides were selected as surrogate peptides for quantitation, considering the oxidizable nature of tyrosine. Our data showed that the results for surrogate peptide quantity measured by our method and by traditional isotope dilution method are in excellent agreement, with the discrepancy of 0.3-3%, validating our CMS method for absolute quantitation. Furthermore, therapeutic monoclonal antibody (mAb) could be quantified by our method as well. Due to the high specificity and sensitivity of MS and no need to use isotope-labeled peptide standards, our CMS method would be of high value for the absolute proteomic quantification.

Online Monitoring of Methanol Electro-Oxidation Reactions by Ambient Mass Spectrometry.

Online detection of methanol electro-oxidation reaction products [e.g., formaldehyde (HCHO)] by mass spectrometry (MS) is challenging, owing to the high salt content and extreme pH of the electrolyte solution as well as the difficulty in ionizing the reaction products. Herein we present an online ambient mass spectrometric approach for analyzing HCHO generated from methanol electro-oxidation, taking the advantage of high salt tolerance of desorption electrospray ionization mass spectrometry (DESI-MS). It was found that HCHO can be detected as PhNHNH+=CH2 (m/z 121) by DESI after online derivatization with PhNHNH2. With this approach, the analysis of HCHO from methanol electro-oxidation by MS was carried out not only in acidic condition but also in alkaline media for the first time. Efficiencies of different electrodes for methanol oxidation at different pHs were also evaluated. Our results show that Au electrode produces more HCHO than Pt-based electrodes at alkaline pH, while the latter have higher yields at acidic solution. The presented methodology would be of great value for elucidating fuel cell reaction mechanisms and for screening ideal fuel cell electrode materials. Graphical Abstract ᅟ.

Coupling Electrochemistry with Probe Electrospray Ionization Mass Spectrometry.

A new coupling of electrochemistry with mass spectrometry (MS) using probe electrospray ionization (PESI) is presented. Due to the high salt tolerance of PESI, the detection of electrochemical reaction products in room-temperature ionic liquids (RTILs) is realized for the first time. Furthermore, PESI-MS allows the analysis of electrochemical reaction products on different or multiple electrode surfaces. In addition, peptides and proteins fractionated through isoelectric focusing (IEF) in the presence of an external electric field can also be directly analyzed by using PESI-MS, suggesting a new and rapid characterization means for the IEF technique. This study reveals the versatility of EC/PESI-MS, which could have an impact in electrochemistry and bioanalysis fields.

Online Investigation of Aqueous-Phase Electrochemical Reactions by Desorption Electrospray Ionization Mass Spectrometry.

Electrochemistry (EC) combined with mass spectrometry (MS) is a powerful tool for elucidation of electrochemical reaction mechanisms. However, direct online analysis of electrochemical reaction in aqueous phase was rarely explored. This paper presents the online investigation of several electrochemical reactions with biological relevance in the aqueous phase, such as nitrosothiol reduction, carbohydrate oxidation, and carbamazepine oxidation using desorption electrospray ionization mass spectrometry (DESI-MS). It was found that electroreduction of nitrosothiols [e.g., nitrosylated insulin B (13-23)] leads to free thiols by loss of NO, as confirmed by online MS analysis for the first time. The characteristic mass shift of 29 Da and the reduced intensity provide a quick way to identify nitrosylated species. Equally importantly, upon collision-induced dissociation (CID), the reduced peptide ion produces more fragment ions than its nitrosylated precursor ion (presumably the backbone fragmentation cannot compete with the facile NO loss for the precursor ion), thus facilitating peptide sequencing. In the case of saccharide oxidation, it was found that glucose undergoes electro-oxidation to produce gluconic acid at alkaline pH, but not at neutral and acidic pHs. Such a pH-dependent electrochemical behavior was also observed for disaccharides such as maltose and cellobiose. Upon electrochemical oxidation, carbamazepine was found to undergo ring contraction and amide bond cleavage, which parallels the oxidative metabolism observed for this drug in leucocytes. The mechanistic information of these redox reactions revealed by EC/DESI-MS would be of value in nitroso-proteome research and carbohydrate/drug metabolic studies.

Recent advances of electrochemical mass spectrometry.

This review article surveys some recent developments of electrochemistry (EC) in combination with mass spectrometry (MS) including instrumentation and bioanalytical applications.

Study of electrochemical reactions using nanospray desorption electrospray ionization mass spectrometry.

The combination of electrochemistry (EC) and mass spectrometry (MS) is a powerful analytical tool for studying mechanisms of redox reactions, identification of products and intermediates, and online derivatization/recognition of analytes. This work reports a new coupling interface for EC/MS by employing nanospray desorption electrospray ionization, a recently developed ambient ionization method. We demonstrate online coupling of nanospray desorption electrospray ionization MS with a traditional electrochemical flow cell, in which the electrolyzed solution emanating from the cell is ionized by nanospray desorption electrospray ionization for MS analysis. Furthermore, we show first coupling of nanospray desorption electrospray ionization MS with an interdigitated array (IDA) electrode enabling chemical analysis of electrolyzed samples directly from electrode surfaces. Because of its inherent sensitivity, nanospray desorption electrospray ionization enables chemical analysis of small volumes and concentrations of sample solution. Specifically, good-quality signal of dopamine and its oxidized form, dopamine o-quinone, was obtained using 10 µL of 1 µM solution of dopamine on the IDA. Oxidation of dopamine, reduction of benzodiazepines, and electrochemical derivatization of thiol groups were used to demonstrate the performance of the technique. Our results show the potential of nanospray desorption electrospray ionization as a novel interface for electrochemical mass spectrometry research.

Electrochemistry-assisted top-down characterization of disulfide-containing proteins.

Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with a top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then undergo online ionization into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows one to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by ß-lactoglobulin A (24 vs 75 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research.

Coupling of liquid chromatography with mass spectrometry by desorption electrospray ionization (DESI).

A liquid chromatography/mass spectrometry (LC/MS) method using desorption electrospray ionization (DESI) as a versatile interface has been established, which allows a wide range of elution flow rates, online derivatization via reactive DESI and further combination with electrochemistry.

Online mass spectrometric analysis of proteins/peptides following electrolytic cleavage of disulfide bonds.

The disulfide bond bridge is an important post-translational modification for proteins. This study presents a structural analysis of biologically active peptides and proteins containing disulfide bonds using electrochemistry (EC) online combined with desorption electrospray ionization mass spectrometry (DESI-MS), in which the sample undergoes electrolytic disulfide cleavage in an electrochemical flow cell followed by MS detection. Using this EC/DESI-MS method, the disulfide-containing peptides can be quickly identified from enzymatic digestion mixtures, simply based on the abrupt decrease in their relative ion abundances after electrolysis. Peptide mass mapping and tandem MS analysis of the ions of the resulting free peptide chains can possibly establish the disulfide linkage pattern and sequence the precursor peptides. In this regard, the method provides much more chemical information than previous analogous electrochemical analyses. In addition, derivatization of thiols by selective selenamide reagents is useful for easy recognition of reduced peptide ions and the number of their free thiols. Furthermore, electrolytic reduction of proteins (e.g., α-lactalbumin) leads to increased charges on the detected protein ions, revealing the role of disulfide bonds on maintaining protein conformation. This electrochemical mass spectrometric method is fast (completed in few minutes) and does not need chemical reductants, potentially having valuable applications in proteomics research.

Online coupling of electrochemical reactions with liquid sample desorption electrospray ionization-mass spectrometry.

The combination of electrochemistry (EC) and mass spectrometry (MS) is a powerful analytical tool to study redox reactions. This work reports the online coupling of a thin-layer electrochemical flow cell with liquid sample desorption electrospray ionization mass spectrometry (DESI-MS) and its applications in investigating various electrochemical reactions of biological molecules such as oxidative formation and reductive cleavage of disulfide bonds and online derivatization of peptides/proteins. As a result of the direct sampling nature of DESI, several useful features of such a coupling have been found, including simple instrumentation, fast response time (e.g., 3.6 s in the case of dopamine oxidation), freedom to choose a favorable ionization mode of DESI or traditional electrolysis solvent systems, and the absence of background signal possibly resulting from ionization when the cell is off (e.g., in the case of dopamine oxidation). More importantly, with the use of this new coupling apparatus, three disulfide bonds of insulin were fully cleaved by electrolytic reduction and both the A and B chains of the protein were successfully detected online by DESI-MS. In addition, online tagging of free cysteine residues of peptides/proteins employing electrogenerated dopamine o-quinone can be performed. These revealed characteristics of the coupling along with examined electrochemical reactions suggest that EC/DESI-MS has good potential in bioanalysis.