Aqueous microdroplets enable abiotic synthesis and chain extension of unique peptide isomers from free amino acids.

Amide bond formation, the essential condensation reaction underlying peptide synthesis, is hindered in aqueous systems by the thermodynamic constraints associated with dehydration. This represents a key difficulty for the widely held view that prebiotic chemical evolution leading to the formation of the first biomolecules occurred in an oceanic environment. Recent evidence for the acceleration of chemical reactions at droplet interfaces led us to explore aqueous amino acid droplet chemistry. We report the formation of dipeptide isomer ions from free glycine or L-alanine at the air-water interface of aqueous microdroplets emanating from a single spray source (with or without applied potential) during their flight toward the inlet of a mass spectrometer. The proposed isomeric dipeptide ion is an oxazolidinone that takes fully covalent and ion-neutral complex forms. This structure is consistent with observed fragmentation patterns and its conversion to authentic dipeptide ions upon gentle collisions and for its formation from authentic dipeptides at ultra-low concentrations. It also rationalizes the results of droplet fusion experiments that show that the dipeptide isomer facilitates additional amide bond formation events, yielding authentic tri- through hexapeptides. We propose that the interface of aqueous microdroplets serves as a drying surface that shifts the equilibrium between free amino acids in favor of dehydration via stabilization of the dipeptide isomers. These findings offer a possible solution to the water paradox of biopolymer synthesis in prebiotic chemistry.

Two-Dimensional Tandem Mass Spectrometry for Biopolymer Structural Analysis.

Biopolymer analysis, including proteomics and glycomics, relies heavily on the use of mass spectrometry for structural elucidation, including sequence determination. Novel methods to improve sample workup, instrument performance, and data analysis continue to be developed to address shortcomings associated with sample preparation, analysis time, data quality, and data interpretation. Here, we present a new method that couples in-source collision-induced dissociation (IS-CID) with two-dimensional tandem mass spectrometry (2D MS/MS) as a way to simplify proteomics and glycomics workflows while also providing additional insight into analyte structures over traditional MS/MS experiments. Specifically, IS-CID is employed as a gas-phase digestion method, i.e., to break down intact full-length polysaccharide or peptide ions prior to mass analysis. The resulting mixtures of oligomeric ions are analyzed by 2D-MS/MS, a technique that allows association of product ions with their precursor ions without isolation of the latter. A novel data analysis strategy is introduced to leverage the second dimension of 2D MS/MS spectra, in which stairstep patterns, representing outputs of a molecule's MSn scans, are extracted for structural interconnectivity information on the oligomer. The results demonstrate the potential applicability of 2D MS/MS strategies to the modern omics workflow and structural analysis of various classes of biopolymers.

PFAS Electroanalysis in Low-Oxygen River Water Using Electrogenerated Dioxygen.

Per- and polyfluoroalkyl substances (PFAS), nicknamed "forever chemicals" due to the strength of their carbon-fluorine bonds, are a class of potent micropollutants that cause deleterious health effects in mammals. The current state-of-the-art detection method requires the collection and transport of water samples to a centralized facility where chromatography and mass spectrometry are performed for the separation, identification, and quantification of PFAS. However, for efficient remediation efforts to be properly informed, a more rapid in-field testing method is required. We previously demonstrated the development and use of dioxygen as the mediator molecule. The use of dioxygen is predicated on the assumption that there will be consistent ambient dioxygen levels in natural waters. This is not always the case in hypoxic groundwater and at high altitudes. To overcome this challenge and further advance the strategies that will enable in-field electroanalysis of PFAS, we demonstrate, as a proof of concept, that dioxygen can be generated in solution through the hydrolysis of water. The electrogenerated dioxygen can then be used as a mediator molecule for the indirect detection of PFOS via molecularly imprinted polymer (MIP)-based electroanalysis. We demonstrate that calibration curves can be constructed with high precision and sensitivity (LOD < 1 ppt or 1 ng/L). Our results provide a foundation for enabling in-field hypoxic PFAS electroanalysis.

2D MS/MS Spectra Recorded in the Time Domain Using Repetitive Frequency Sweeps in Linear Quadrupole Ion Traps.

Ion trap mass spectrometers have emerged as powerful on-site analytical platforms, in spite of limited mass resolution, due to their compatibility with ambient ionization methods and ready implementation of tandem mass spectrometry (MS/MS). When operated at constant trapping voltage, ions can be activated at their secular frequencies and all MS/MS experiments can be performed, including the two-dimensional tandem mass scan (2D MS/MS scan) in which all precursor ions and their subsequent product ions are both identified and correlated. In the new method of performing this 2D MS/MS experiment presented here, the precursor ions are excited by a nonlinear (inverse Mathieu q) frequency sweep while the resulting product ions are identified by their ejection time within a repeating orthogonally applied nonlinear (inverse Mathieu q) frequency sweep. This resulting compact representation contains the total fragmentation behavior of a collection of ionized compounds and captures detailed chemical information efficiently (typically in 1 s). The approach is implemented using a simple single mass analyzer instrument. This methodology was tested on three different multicomponent mixtures: drugs of abuse, peptides, and fentanyl analogs. The data are compared with those obtained by more common MS/MS scan methods.

High-Throughput Label-Free Enzymatic Assays Using Desorption Electrospray-Ionization Mass Spectrometry.

High-throughput (HT) enzymatic assays, which typically rely on labeled compounds and plate readers, are important for drug discovery. Mass spectrometry (MS) provides an alternative method of performing HT label-free assays. Here we demonstrate the use of a HT platform based on desorption electrospray ionization (DESI) MS for the label-free study of enzymatic reactions directly from the bioassay matrix with an effective analysis time of 0.3 s per sample. This system allows for thorough analysis of the enzymatic process through monitoring of its substrate and product after an external calibration. We show the platform capabilities by an in-depth study of the acetylcholinesterase assay, including kinetic parameter determination, rapid inhibitor screening, and further characterization of positive hits (that is, IC50 and Ki ), as well as inhibition-reactivation assays. We anticipate that the expansion of this platform has high potential impact in label-free enzymology as well as in drug discovery.

Breaking down microdroplet chemistry.

Charged microdroplets accelerate mineral disintegration.

Reaction acceleration at the surface of a levitated droplet by vapor dosing from a partner droplet.

Chemical reactions in micrometer-sized droplets can be accelerated by up to six orders of magnitude. However, this acceleration factor (ratio of rate constants relative to bulk) drops to less than 10 for millimeter-sized droplets due to the reduction in surface/volume ratio. To enhance the acceleration in millimeter-sized droplets, we use a new synthesis platform that directly doses reagent vapor onto the reaction droplet surface from a second levitated droplet. Using Katritzky transamination as a model reaction, we made quantitative measurements on size-controlled vapor-dosed droplets, revealing a 31-fold increase in reaction rate constants when examining the entire droplet contents. This enhancement is attributed to a greater reaction rate constant in the droplet surface region (estimated as 105 times greater than that for the bulk). The capability for substantial reaction acceleration in large droplets highlights the potential for rapid synthesis of important chemicals at useful scales. For example, we successfully prepared 23 pyridinium salts within minutes. This efficiency positions droplets as an exceptional platform for rapid, in situ catalyst synthesis. This is illustrated by the preparation of pyridinium salts as photocatalysts and their subsequent use in mediation of amine oxidation both within the same droplet.

Automated High-Throughput System Combining Small-Scale Synthesis with Bioassays and Reaction Screening.

The Purdue Make It system is a unique automated platform capable of small-scale in situ synthesis, screening small-molecule reactions, and performing direct label-free bioassays. The platform is based on desorption electrospray ionization (DESI), an ambient ionization method that allows for minimal sample workup and is capable of accelerating reactions in secondary droplets, thus conferring unique advantages compared with other high-throughput screening technologies. By combining DESI with liquid handling robotics, the system achieves throughputs of more than 1 sample/s, handling up to 6144 samples in a single run. As little as 100 fmol/spot of analyte is required to perform both initial analysis by mass spectrometry (MS) and further MSn structural characterization. The data obtained are processed using custom software so that results are easily visualized as interactive heatmaps of reaction plates based on the peak intensities of m/z values of interest. In this paper, we review the system's capabilities as described in previous publications and demonstrate its utilization in two new high-throughput campaigns: (1) the screening of 188 unique combinatorial reactions (24 reaction types, 188 unique reaction mixtures) to determine reactivity trends and (2) label-free studies of the nicotinamide N-methyltransferase enzyme directly from the bioassay buffer. The system's versatility holds promise for several future directions, including the collection of secondary droplets containing the products from successful reaction screening measurements, the development of machine learning algorithms using data collected from compound library screening, and the adaption of a variety of relevant bioassays to high-throughput MS.

High-yield gram-scale organic synthesis using accelerated microdroplet/thin film reactions with solvent recycling.

A closed system has been designed to perform microdroplet/thin film reactions with solvent recycling capabilities for gram-scale chemical synthesis. Claisen-Schmidt, Schiff base, Katritzky and Suzuki coupling reactions show acceleration factors relative to bulk of 15 to 7700 times in this droplet spray system. These values are much larger than those reported previously for the same reactions in microdroplet/thin film reaction systems. The solvent recycling mode of the new system significantly improves the reaction yield, especially for reactions with smaller reaction acceleration factors. The microdroplet/thin film reaction yield improved on recycling from 33% to 86% and from 32% to 72% for the Katritzky and Suzuki coupling reactions, respectively. The Claisen-Schmidt reaction was chosen to test the capability of this system in gram scale syntheses and rates of 3.18 g per h and an isolated yield of 87% were achieved.