High-throughput quantitation of large molecules using multiplexed chromatography and high-resolution/accurate mass LC-MS

Background: High-throughput screening with LC-MS has been routinely implemented to various degrees throughout the entire drug-discovery process. One of the major advantages of utilizing LC-MS earlier at the lead discovery stage is reducing the cost of sample analysis while increasing assay selectivity. Avoiding labeling agents and other non-native species in an assay environment can reduce costly sample preparation, while chromatographic separation of the analyte of interest from interferences in the sample matrix has been shown to increase selectivity and sensitivity. Methods: In this paper, we utilize high-resolution MS-LC multiplexing to analyze phosphorylated peptides as part of a screening assay. Commonly used enzyme buffers were used to prepare phosphorylated peptide standards of varying concentrations and these were plated into a 96-well plate format for LC-MS analysis. The overall cycle time for analysis from sample to sample, LLOQ, Z' and coefficient of variance were determined. Conclusion: High-resolution MS coupled with LC multiplexing provides high-quality sample analysis at sampling rates of up to 18 s per sample. Samples analyzed in both simple and complex sample matrixes demonstrated an LOQ of 5 nM with linear response across the working range of the assay. Overall statistical analysis of the large samples produced Z' = 0.85 for sample sets in sodium citrate solution and Z' = 0.66 for sample sets in HEPES solution indicating a robust analytical method.