- Triple Sharp Venture Engineering
Accelerating the drug discovery process is a key priority for pharma-ceutical businesses around the world and the initial screening of very large compound libraries remains a critical first step. New technology and new applications of methods proven in other areas of research are constantly being assessed by discovery scientists. Everyone is looking for something to give them a competitive edge – more information, faster screening or deeper understanding.
In this article, we explore the significant potential of mass spectrometry methodology to add value in the drug discovery process, and look specifically at how the latest matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) systems can boost analysis speed and increase productivity, while reducing costs and waste in high throughput screening (HTS) applications.
Why MALDI-TOF?
Traditional biochemical HTS assays are generally based on color-imetric or fluorescence detection methods, such as time-resolved fluorescence energy transfer and fluorescence intensity. They enable high throughput – more than 50,000 samples can be processed per day – but they are single point, single analyte methods that call for chemical derivatization of one or more of the labelling reagents needed for detection.
As a result, label-free methods based on mass spectrometry (MS) have rapidly expanded into the HTS environment. Initially, these MS-based methods used multiplexed liquid chromatography (LC)-MS technology. This approach provides significantly more information on each sample; however, with typical cycle times of up to 30 seconds per sample, daily throughput is limited. Solid Phase Extraction (SPE) based techniques that are capable of analyzing samples in around 10 seconds have also been introduced. Despite being dramatically faster than LC-MS methods, an analysis time of 10 seconds is still considered relatively slow for routine HTS.
Consequently, the drive to find a new approach continued and much has been written about the factors to look for when assessing an ideal HTS technology (Table 1).
Table 1.
A New Benchmark: Results from a Recent Case Study
Recent work has established a new performance benchmark for an optimized, fully automated MALDI-TOF system (Figure 1).
Method
Quenched assay reaction mixture plates, alpha-cyano-4- hydroxycinnamic acid matrix and wash containers were introduced to a fully automated robotic system (MALDI PharmaPulse, Bruker Inc.). The system included an ACell robotic arm (HighRes Biosolutions), a LidValet de-lidder (HighRes Biosolutions), a plate hotel and mosquito HTS liquid handler (TTP Labtech).
The system transferred, in 1536 well format, a sample of reaction mixture and matrix onto MALDI targets comprised of an adapter and polished steel insert with 1.0 mm thickness.
After drying, the samples were analyzed on an autoflex speed MALDI-TOF mass spectrometer (Bruker Inc.) equipped with a 2 kHz solid state laser and Acquiris 4 GHz digitizer.
The automated runs were set up with 100 - 1000 laser shots per spectrum to assess the overall speed of analysis.
The run time per target was averaged from three measurements and used to calculate the analysis time per sample and the throughput per day.
Results
The analysis time of MALDI high-throughput screening runs was consistently less than 1 second per sample, with best performance at a significantly reduced to 0.54 seconds per sample (Table 3). This equates to an ability to analyze more than 121,000 samples per day.
Conclusion
MALDI PharmaPulse achieved speeds of 0.5-1.0 seconds per sample.
MALDI-HTS throughput of more than 100,000 samples per day is equivalent to the rate offered by traditional fluorescence based screening methods, while also enabling multiplexed detection of any number of products, side products and contaminants.
Most recently, MALDI-TOF has been revisited and has now emerged as a powerful alternative for high throughput MS detection in the pharmaceutical industry. Compared to the alternative LC-MS and SPEMS methods, MALDI-TOF systems offer:
- True label-free detection of native substrates and products in functional biochemical assays
- Fast MS-based screening with cycle times of less than 1s per sample
- Reduced sample volume (typically 250 nL)
- Significantly reduced solvent use and subsequent chemical waste
- Straightforward method development, with no sample pre-treatment required
- Robust automation options
Predicting Real-World Performance
To translate these features into real-world performance advantages to facilitate the drug discovery process, we could consider the time needed to complete a screen of 200,000 compounds. Table 2 compares a x4 multiplex assay with an assumed cycle time of 20 seconds per sample, an SPE-MS method with a cycle time of 7.5 seconds per sample, and a MALDI-TOF protocol with a 1 second cycle time per sample.
Table 2.
Using MALDI-TOF, the screen would be completed in 3 days. The SPE-MS method would require 20 days, and the multiplex assay would take 54 days.
Looking Ahead
An objective assessment of MADLI-TOF reveals that the technique ticks all the boxes for a new HTS methodology with the pharmaceutical setting. It enables the monitoring of relevant products; assays have sensitivity in the range 10-100 nM, ideal for most analytes; and methods are straightforward to develop. Instruments are robust in use and are amenable to standard automation approaches. Moreover, assays are low cost, compared to the alternative LCMS and SPE-MS methods.
Perhaps most importantly, MALDI-TOF now offers the speed that is needed to screen the very largest libraries of compounds. A genuine acceleration of the vital early stages of drug discovery has been realized.