Development of IR-MALDESI-MS for High Throughput Biochemical, Cellular, and Protein Assays

High-throughput screening (HTS) is crucial to generate hits and leads in drug discovery efforts. Most high-throughput biochemical assays are based on optical detection or radioactivity with high sensitivity and a detection speed of sub-second per well. These assays usually require labeling of substrates/products or coupling reactions. False positives for optical assays and material handling for radioactivity assays can lead to detection artifacts, increase cost, and delay progress in a drug discovery program. Considering the benefits of specificity that MS-based HTS assays offer, there is increased interest in recent years to develop alternative strategies to improve throughput using robust chromatography-free MS methods. Examples of such implementations include the use of matrix-assisted laser desorption ionization (MALDI), solid phase extraction (SPE)-based RapidFire system and ambient ionization techniques such as acoustic droplet ejection and acoustic mist ionization (AMI).

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid ambient ionization method that combines the use of laser desorption and electrospray ionization (ESI). Originally developed for tissue imaging, it has significant potential for high throughput analysis of biological samples. Unlike MALDI, the matrix required for IR-MALDESI is water or ice, so no extra sample preparation is necessary for biological samples such as tissue, biofluids or buffered biochemical reactions. Decoupling the laser desorption process and allowing for post-desorption electrospray ionization limits ion suppression from salts and detergents, common additives for biochemical assay buffer systems, allowing biologically relevant buffers to be used with little concession to MS compatibility. Additionally, the theoretical limit on sampling rate is very high since laser desorption, electrospray ionization, and introduction of sample to the MS are achieved on the millisecond timescale, shifting the rate limiting steps to stage movement and MS data acquisition speed, which varies depending upon the mass analyzer type and operation mode. In addition to speed and compatibility with biological matrices IR-MALDESI can sample from any standard plate type and the cost of the ionization source (laser, stage, ESI source, etc.) is relatively inexpensive. This minimizes both consumable and capital expenses.

This talk will describe the implementation and optimization of IR-MALDESI for high throughput biochemical and cellular assays as well as high throughput analysis of protein post-translational modifications and protein-compound interactions. By coupling an IR-MALDESI ionization source to a Q Exactive HF-X mass spectrometer we have been able to achieve a 22 Hz sampling rate for biochemical and cellular assays while maintaining good quality assay Z’ of > 0.5 and %CV < 10%. In addition, whole proteins, up to 150 kDa, can be analyzed in < 1s and post-translational modifications or covalent compound additions monitored. Limited top-down sequencing of modification sites can also be performed on the Q Exactive instrument in short timeframes. Examples of each of these applications will be shown. In addition to the examples shown IR-MALDESI has potential to significantly increase productivity for ADME assays, PK/PD samples, permeability measurements, affinity-selection mass spectrometry, fermentation monitoring and multiple other applications in drug discovery. Its original develoment as an MS imaging technique also allows it to be used for imaging and solid surface analysis such as tablet degradation analysis, product QC, or counterfeit testing. 

Learning Objectives:

1. List three characteristics of an ideal high throughput assay detection technology.

2. Compare and contrast MALDESI-MS with LC-MS in terms of speed, versatility and sensitivity.

3. Discuss what experiments you could conduct with access to a MALDESI system.