Overcoming a false-positive mechanism in RapidFire MRM-based high throughput screening.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Drug Discovery

Background:

• High-throughput screening (HTS) is crucial for drug discovery but is often hampered by false-positive results, leading to wasted resources.
• Mass spectrometry (MS) offers a direct detection method for enzyme reaction products in HTS, avoiding artifacts common in other assay types.

Purpose of the Study:

• To report a previously undescribed mechanism causing false-positive hits in MS-based HTS.
• To develop a pipeline for identifying these specific false-positive hits.
• To propose a method for mitigating these false positives.

Main Methods:

• Utilized mass spectrometry for direct detection of enzyme reaction products in a screening assay.
• Developed and implemented a computational pipeline to analyze screening data for a specific false-positive signature.
• Designed and tested a mitigation strategy to reduce the occurrence of identified false positives.

Main Results:

• Identified a novel mechanism contributing to false-positive results in MS-based HTS.
• Successfully developed a pipeline capable of detecting these specific false-positive hits.
• Demonstrated the effectiveness of a proposed method in mitigating these false positives.

Conclusions:

• A new source of artifacts in MS-based HTS has been characterized.
• The developed detection pipeline and mitigation strategy can improve the accuracy and efficiency of drug discovery screening.
• Implementing these methods can reduce resource expenditure by minimizing false-positive compound identification.