Ultra-high-throughput SPE-MALDI workflow: Blueprint for efficient purification and screening of peptide libraries

Highlights: • mRNA display peptides require ultra-high throughput SPE-MALDI purification to avoid bottlenecks. • SPE purification is possible by scaling out automation-ready robotic workflows. • uHT-MALDI facilitates synthetic hit detection on a 384-well plate within 30 minutes. • uHT-SPE-MALDI outpaces conventional HPLC-DAD-MS while reducing environmental impact. At the forefront of synthetic endeavors in the pharmaceutical industry, including drug discovery and high-throughput screening, timelines are tight and large quantities of pure chemical targets are rarely available. In this regard, the development of novel and increasingly challenging chemistries requires a commensurate level of innovation to develop reliable analytical assays and purification workflows with rapid turnaround that enables accelerated pharmacological evaluation. A small-scale automation platform enabling high-throughput analysis and purification to streamline the selection of candidate leads would be a transformative advance. Herein, we introduce an automation-friendly solid-phase extraction-matrix-assisted laser desorption/ionization (SPE-MALDI) platform applied to the high-throughput purification and analysis of peptide libraries. This advance enabled us to purify peptides from microgram levels in less than a day with results comparable to traditional high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS).