Ultrafast peptide preparation brings shotgun proteomics into the minute era

Abstract

Background

Mass spectrometry-based shotgun proteomics relies on efficient sample preparation, where proteins are reduced, alkylated, and digested into peptides for LC-MS/MS analysis. While LC-MS/MS and bioinformatic identification have advanced to minute-scale workflows, sample preparation remains a major bottleneck, typically requiring hours to days. Although methods such as high-pressure and droplet-based reactions have reduced processing times, achieving minute-scale preparation has remained challenging. There is a clear need for an integrated, rapid sample preparation strategy to match the speed of modern LC-MS/MS and data analysis platforms.

Results

We developed OPPRAD (One-Pot Protein Reduction, Alkylation, and Digestion), an ultrafast sample preparation method conducted in water-in-oil nano/micro-droplets. This one-pot process completes all three key reactions within 5 min. When integrated with rapid LC-MS/MS on the Orbitrap Astral platform and MSFragger- or DIA–NN–based identification, the entire workflow achieved serum-to-peptide identification in 25.9 min and tissue-to-identification in 35.9 min—the fastest reported proteomic pipeline. The method showed high peptide recovery (∼68–71%), excellent cysteine alkylation (>99%), and miss cleavage rates comparable to conventional bulk digestion. It enabled deep proteome coverage across a wide dynamic range and was successfully applied to identify differentially expressed proteins in paired liver cancer tissues.

Significance

OPPRAD drastically shortens proteomic sample preparation from hours to minutes, reducing steps, cost, and hands-on time. This innovation bridges a critical gap in high-throughput proteomics, enabling rapid translational applications and large-scale clinical studies. The method's speed and efficiency pave the way for real-time proteomic analysis and future integration with online desalting and database searching.