Label-Free High-Throughput Screening via Mass Spectrometry: A Single Cystathionine Quantitative Method for Multiple Applications

Abstract

Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. MS permits the selective detection of enzyme substrates or products in a wide range of biological matrices without need for derivatization, labeling, or capture technologies. As part of a cardiovascular drug discovery effort aimed at finding modulators of cystathionine beta-synthase (CBS), we used the RapidFire((R)) label-free high-throughput MS (HTMS) technology to develop a high-throughput screening (HTS) assay for CBS activity. The in vitro assay used HTMS to quantify the unlabeled product of the CBS reaction, cystathionine. Cystathionine HTMS analyses were carried out with a throughput of 7 s per sample and quantitation over a linear range of 80-10,000 nM. A compound library of 25,559 samples (or 80 384-well plates) was screened as singlets using the HTMS assay in a period of 8 days. With a hit rate of 0.32%, the actives showed a 90% confirmation rate. The in vitro assay was applied to secondary screens in more complex matrices with no additional analytical development. Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project.

Establishment of glycosaminoglycan assays for mucopolysaccharidoses

“…The speed and efficiency of the HT-MS/MS can allow for experiments that would otherwise be deemed "untenable" under normal circumstances. The HT system has been validated as suitable for many drug discoveries [58][59][60][61][62][63][64][65] and ADME (Absorption, Distribution, Metabolism and Excretion) based applications [66]. The main drawback of this method is that disaccharides with identical molecular weights cannot be distinguished.…”

Measurement of glycolysis reactants by high-throughput solid phase extraction with tandem mass spectrometry: Characterization of pyrophosphate-dependent phosphofructokinase as a case study

“…The MS-based analysis of multienzyme glycolytic systems has been described previously [28], further suggesting that high-throughput SPE-MS/MS could be instrumental in simultaneously studying multiple enzymes within a complex network. Because SPE-MS/MS can accommodate a variety of packing materials, can be run in reverse of normal phase, and can be connected to any mass spectrometer, this detection strategy facilitates the measurement of a wide breadth of analytes including small molecules [29][30][31][32][33][34], peptides [35][36][37], and whole proteins [38]. This work, therefore, also contributes to a growing list of applications for high-throughput SPE-MS/MS analysis.…”

Fast and Reliable CYP Inhibition Assays

“…Recently, a device called " RapidFire " was introduced that combines sampling in 96 -or 384 -well plates, online SPE, and quantifi cation by a mass spectrometer that reached analytical speed at 5 s per injection (Holt et al, 2009 ). The HLM SPE -MS assays greatly enhanced throughput of metabolite quantifi cation and produce the same quality of result as HLM -LC -MS inhibition assays.…”