Peptide and protein aggregation is a hallmark of numerous neurodegenerative diseases. However, the structural characterization of oligomeric intermediates remains challenging due to their low abundance, heterogenic nature, and the complexity of coinciding and transient reaction networks. Confining reactions to microenvironments, such as picoliter droplets in droplet-based microfluidics (DBMF), simplifies these networks and thereby enabling high-throughput analysis and controlled reaction kinetics. This study explores the integration of DBMF with ion mobility mass spectrometry (IM-MS) to probe aggregation kinetics in droplets. We address key technical challenges, including interference from the dispersed phase, surfactant, and DBMF device material, and we present optimization strategies for droplet generation, and surfactant concentration, to reduce contamination. Furthermore, we investigate instrumentation-based influences on ionization to improve signal stability. Using model peptides, bradykinin, leucine-enkephalin, and the aggregation-prone segment Ac-PHF6-NH₂ from the tau protein, we demonstrate the successful development of a sensitive method. Additionally, using the Ac-PHF6-NH₂ peptide, oligomeric transient species were probed and characterized by their IM and m/z values. This work demonstrates the potential of DBMF-IM-MS to study peptide aggregation in confined environments, providing insights into the formation of oligomeric species and laying the groundwork for studies on larger proteins and the development of therapeutic agents targeting aggregation using multi-channel devices.

Ambient ionization coupled to mass spectrometry has the advantages of minimal requirements for sample preparation prior to analysis which renders it suitable for high throughput screening. We present a protocol that permits the application of this method in routine biotechnology and chemical biology laboratories which are using engineered enzymes to produce target compounds from substrates. We show how DESI-MS can be used to directly analyse the activity of biotransformations from crude cell lysate which we term DiBT-MS, this method is 10-1000 times faster than LC-MS and uses far less solvent. This protocol demonstrates the impact of solvent spray composition on ionization efficiency of the target analyte, the benefits of a nylon membrane slide and the reusability of sample slides in multiple experiments.

Droplet microfluidics coupled with mass spectrometry is evolving for high throughput screening. A commercial solution that can readily interface to existing mass spectrometry instrumentation would be highly desirable and allow the technology to become accessible to those without experience in droplet microfluidics. Here, we demonstrate a droplet reinjection workflow to infuse samples into three common mass spectrometers, each with different electrospray ionisation (ESI) source configurations. The ease of chip-mass spectrometry (MS) coupling is explored and difficulties highlighted. Particular attention has been turned to ESI sources which apply the electrospray voltage directly to the incoming droplet emitter (‘push source’), as these were found to cause droplet coalescence within the microfluidic chip reinjection channel. To overcome such a difficulty, we have identified different solutions including a grounded mesh insert as a shielding approach, and minor changes to chips and ESI sources to stabilise incoming droplets. Results obtained allow the mass spectrum of individual reinjected droplets to be determined without hindrance from the electrospray voltage. The problems and solutions identified here, outline the foundations for the development of a commercial droplet reinjection-mass spectrometry interface.

The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical timescale. Here we show the application of direct infusion mass spectrometry (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ~40 seconds per sample. The heat-map output allows rapid selection of active enzymes within 96-well plates facilitating identification of industrially relevant biocatalysts. This DiBT-MS screening workflow has been applied to the directed evolution of a phenylalanine ammonia lyase (PAL), enhancing its activity towards electron-rich cinnamic acid derivatives which are relevant to lignocellulosic biomass degradation. Additional benefits of the screening platform include the discovery of biocatalysts (kinases, imine reductases) with novel activities and the incorporation of ion mobility technology for the identification of product hits with increased confidence.

High and ultra-high-throughput label-free sample analysis is required by many applications, extending from environ-mental monitoring to drug discovery and industrial biotechnology. HTS methods predominantly are based on a targeted workflow, which can limit their scope. Mass spectrometry readily provides chemical identity and abundance for complex mixtures and here, we use microdroplet generation microfluidics to supply picolitre aliquots for analysis at rates up to and including 33 Hz. This is demon-strated for small molecules, peptides and proteins up to 66 kDa on three commercially available mass spectrometers from salty solutions to mimic cellular environments. Designs for chip-based interfaces that permit this coupling are presented and the merits and challenges of these interfaces are discussed. On an Orbitrap platform droplet infusion rates of 6 Hz are used for the analysis of cytochrome c, on a DTIMS Q-TOF similar rates were obtained and on a TWIMS Q-TOF utilizing IM-MS software rates up to 33 Hz are demonstrated. The potential of this approach is demonstrated with proof of concept experiments on crude mixtures including egg white, unpurified recombinant protein and a biotransformation supernatant.

High throughput screening (HTS) of molecular analytes is in high demand from and implemented in many areas of chemistry, medicine and industrial biotechnology including the discovery of biomarkers and the development of new chemical entities. Despite its prevalence, technical challenges remain in many of the new application areas of HTS which require rapid results from complex mixtures, for example in: screening biotransformations; targeted metabolomics; and in locating drugs and/or metabolites in biological matrices. Common to all of these are lengthy and costly sample preparation stages, involving recovery from cell cultures, extractions followed by low throughput LC-MS/MS methods or specific fluorescence measurements. In the latter the target molecules need to be inherently fluorescent or to include a fluorescent label or tag which can adversely influence a cellular system. Direct infusion mass spectrometry coupled with robotic sample infusion is a viable contender for information rich HTS with sub-second analysis times, and recent developments in ambient ionisation have heralded a new era where screening can be performed on crude cell lysates or even from live cells. Besides commercially available technologies such as RapidFire, Acoustic Mist Ionisation, and the TriVersa ChipMate there are promising new developments from academic groups. Novel applications using desorption electrospray ionisation, microfluidics, rapid LC-separation and 'one cell' direct infusion methods offer much potential for increasing throughput from 'messy' complex samples and for significantly reducing the amount of material that needs to be analysed. Here we review recent advances in HTS coupled with MS with an emphasis on methods that reduce or remove all sample preparation and will facilitate single cell screening approaches.