Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid, ambient ionization source that combines the advantages of electrospray ionization and matrix-assisted laser desorption/ionization, making it a versatile tool for both highthroughput screening (HTS) and mass spectrometry imaging (MSI) studies. To expand the capabilities of the IR-MALDESI source, an entirely new architecture was designed to overcome the key limitations of the previous source. This next-generation (NextGen) IR-MALDESI source features a vertically mounted IR-laser, a planar translation stage with computerized sample height control, an aluminum enclosure, and a novel mass spectrometer interface plate. The NextGen IR-MALDESI source has improved user-friendliness, improved overall versatility, and can be coupled to numerous Orbitrap mass spectrometers to accommodate more research laboratories. In this work, we highlight the benefits of the NextGen IR-MALDESI source as an improved platform for MSI and direct analysis. We also optimize the NextGen MALDESI source component geometries to increase target ion abundances over a wide m/z range. Finally, documentation is provided for each NextGen IR-MALDESI part so that it can be replicated and incorporated into any lab space.

Rationale The development and characterization of the novel NextGen infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) source catalyzed new advancements in IR‐MALDESI instrumentation, including the development of a new analysis geometry. Methods A vertically oriented transmission mode (tm)‐IR‐MALDESI setup was developed and optimized on thawed mouse tissue. In addition, glycerol was introduced as an alternative energy‐absorbing matrix for tm‐IR‐MALDESI because the new geometry does not currently allow for the formation of an ice matrix. The tm geom was evaluated against the optimized standard geometry for the NextGen source in reflection mode (rm). Results It was found that tm‐IR‐MALDESI produces comparable results to rm‐IR‐MALDESI after optimization. The attempt to incorporate glycerol as an alternative matrix provided little improvement to tm‐IR‐MALDESI ion abundances. Conclusions This work has successfully demonstrated the adaptation of the NextGen IR‐MALDESI source through the feasibility of tm‐IR‐MALDESI mass spectrometry imaging on mammalian tissue, expanding future biological applications of the method.

High‐throughput screening (HTS) is a technique mostly used by pharmaceutical companies to rapidly screen multiple libraries of compounds to find drug hits with biological or pharmaceutical activity. Mass spectrometry (MS) has become a popular option for HTS given that it can simultaneously resolve hundreds to thousands of compounds without additional chemical derivatization. For this application, it is convenient to do direct analysis from well plates. Herein, we present the development of an infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) source coupled directly to an Agilent 6545 for direct analysis from well plates. The source is coupled to a quadrupole time‐of‐flight (Q‐TOF) mass spectrometer to take advantage of the high acquisition rates without sacrificing resolving power as required with Orbitrap or Fourier‐transform ion cyclotron resonance (FTICR) instruments. The laser used for this source operates at 100 Hz, firing 1 pulse‐per‐burst, and delivers around 0.7 mJ per pulse. Continuously firing this laser for an extended duration makes it a quasi‐continuous ionization source. Additionally, a metal capillary was constructed to extend the inlet of the mass spectrometer, increase desolvation of electrospray charged droplets, improve ion transmission, and increase sensitivity. Its efficiency was compared with the conventional dielectric glass capillary by measured signal and demonstrated that the metal capillary increased ionization efficiency due to its more uniformly distributed temperature gradient. Finally, we present the functionality of the source by analyzing tune mix directly from well plates. This source is a proof of concept for HTS applications using IR‐MALDESI coupled to a different MS platform.