RapidFire BLAZE-Mode Is Boosting ESI-MS Toward High-Throughput-Screening

Abstract

Label-free in vitro potency assays are an emerging field in drug discovery to enable more physiological conditions, to improve the readout quality, and to save time. For this approach mass spectrometry (MS) is a powerful technology to directly follow physiological processes. The speed of this methodology, however, was for a long time not compatible with chemiluminescence- or fluorescence-based assays. Recent advances in matrix-assisted laser desorption/ionization (MALDI) instrumentation paved the way for high-throughput MS analysis of label-free assays for large compound libraries, whereas electrospray ionization (ESI)-based mass spectrometers equipped with RapidFire autosamplers were limited to medium throughput. Here we present a technological advancement of the RapidFire device to enable cycle times of 2.5 s per sample. This newly developed BLAZE-mode substantially boosted the ESI-MS analysis speed, providing an alternative technology for label-free high-throughput screening.

High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry–based deubiquitylating enzyme assay for drug discovery

“…However, the high sensitivity of the ESI detection to contaminants demands a prior sample cleaning and concentration step that pushes the sample rate in the 7-13 s range 93 , much higher than the~1.5 s currently required for the detection of ubiquitin by the Rapiflex MALDI-TOF mass spectrometer. Recently reported RapidFire BLAZE methodology limits the time of small molecule detection down to 2.5 s per sample 94 , even higher than the MALDI-TOF MS-based detection, but the applicability of such methods to larger molecules has not been assessed.…”

MALDI-TOF-Based Affinity Selection Mass Spectrometry for Automated Screening of Protein–Ligand Interactions at High Throughput

“…Signals of Ref_Cpd. 1 showed a coefficient of variation (CV) of 34.3% around an average AUC of 12,191 (S/N = 593). Normalization of AUC values to the internal standard peptide decreased signal variability to CV = 16.2%, thereby providing an appropriate approach to correct for signal variations attributable to the MALDI process.…”

“…Although technical improvements of ESI instrumentation have shifted analysis cycle times to a few seconds per sample, this throughput is still inadequate for traditional large compound library screens. 12 The abovementioned technologies therefore use compilations of test substances in compound pools comprising up to 2500 members per experiment. 9 While this is a powerful approach for screening specially designed mass-encoded libraries, technical implementation of large-scale compound pooling, complex library deconvolution, and ensuring compound integrity and solubility can be cumbersome.…”

MALDI-TOF Mass Spectrometry-Based High-Throughput Screening for Inhibitors of the Cytosolic DNA Sensor cGAS

“…While recent developments in the field of ESI-based MS suggest promising potential for HTS, MALDI-TOF is the only commercially available MS-based and highthroughput-compatible technology to date. 38,39 The developed cGAS assay was profoundly validated and subsequently used to screen a full-diversity compound library with excellent assay statistics. To the best of our knowledge, this is the first report of a large-scale (>10 6 ) HTS campaign using a MALDI-TOF-based readout strategy.…”