Coupling Droplet Microfluidics with Mass Spectrometry for Ultrahigh-Throughput Analysis of Complex Mixtures up to and above 30 Hz

High- and ultrahigh-throughput label-free sample analysis is required by many applications, extending from environmental 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 picoliter aliquots for analysis at rates up to and including 33 Hz. This is demonstrated 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 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

Rapid screening of diverse biotransformations for enzyme evolution

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 time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s 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) as a case study, enhancing its activity toward 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