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

Area of Science:

• Analytical Chemistry
• Biochemistry
• Drug Discovery

Background:

• Liquid chromatography-mass spectrometry (LC-MS) is vital in drug discovery for cost-effective and selective sample analysis.
• Early-stage LC-MS application enhances assay selectivity and sensitivity by minimizing sample preparation and interference.
• Avoiding labeling agents simplifies assays and reduces costs in lead discovery.

Purpose of the Study:

• To evaluate high-resolution MS-LC multiplexing for analyzing phosphorylated peptides in screening assays.
• To determine key performance metrics including cycle time, lower limit of quantitation (LLOQ), and Z' factor.
• To assess the method's robustness in various sample matrices.

Main Methods:

• Phosphorylated peptide standards were prepared in common enzyme buffers at varying concentrations.
• Samples were analyzed using high-resolution MS coupled with LC multiplexing in a 96-well plate format.
• Assay performance was evaluated by measuring sample-to-sample cycle time, LLOQ, Z' factor, and coefficient of variance.

Main Results:

• High-resolution MS-LC multiplexing achieved sample analysis rates as rapid as 18 seconds per sample.
• The assay demonstrated a lower limit of quantitation (LLOQ) of 5 nM with linear response in both simple and complex matrices.
• Statistical analysis yielded robust Z' values (0.85 in sodium citrate, 0.66 in HEPES), indicating method reliability.

Conclusions:

• High-resolution MS-LC multiplexing offers a robust and efficient analytical method for phosphorylated peptide screening.
• The technique provides high-quality data at accelerated sampling rates, suitable for high-throughput drug discovery.
• The demonstrated LOQ and statistical robustness confirm the method's utility in diverse analytical challenges.