Protein Barcoding and Next-Generation Protein Sequencing Workflow

This document is a preprint article that describes the development of a protein barcoding workflow integrated with single-molecule Next-Generation Protein Sequencing™ (NGPS™) using the benchtop Platinum® instrument. The study outlines the validation of eight peptide barcodes, which are designed to minimize detection bias while maximizing sensitivity across various experimental conditions. The authors detail the optimization of expression constructs to enhance the purification workflow's efficiency. The workflow involves expressing affinity-tagged proteins with unique peptide barcodes, followed by purification and sequencing of these barcodes on the Platinum instrument. The results demonstrate the ability to detect barcodes at 400 fmol concentration in an eight-plex mixture and at 50 fmol for individual barcodes, showcasing a ten-fold dynamic range for low-abundance variants. This approach aims to facilitate future studies on protein interactions and improve targeted drug delivery strategies.