Protein Barcoding and Next-Generation Protein Sequencing for Multiplexed Protein Selection, Analysis, and Tracking

Abstract Protein barcoding has emerged as a powerful tool for the multiplexed identification and characterization of proteins, providing a mechanism for precise tracking of protein affinity, location, and expression. In this study, we describe the development of a protein barcoding workflow for use with single-molecule Next-Generation Protein Sequencing™ (NGPS™) on the benchtop Platinum® and Platinum® Pro instruments. We present data on the validation of eight peptide barcodes, each designed to minimize detection bias and maximize sensitivity across various experimental conditions. We have also optimized the design of expression constructs to decrease both the hands-on time and input requirements of the workflow. In this workflow, affinity-tagged proteins are expressed with unique peptide barcodes. Following experimental selection or treatments, the proteins are purified, and the peptide barcodes are cleaved and sequenced on the Platinum instrument. We demonstrate that we can detect barcodes at 400 fmol of sample input concentration within the eight-plex mixture, and at 50 fmol of sample input for individual barcodes. We also show the capacity of this barcoding approach to achieve a ten-fold dynamic range, underscoring its sensitivity in recovering variants with low abundance. Through the combination of protein barcoding and NGPS, we lay the groundwork for future studies aimed at characterizing protein interactions and improving targeted drug delivery strategies. Competing Interest Statement All authors except GHM are employees and shareholders of Quantum-Si, Inc. Footnotes Added author (GHM) who was inadvertently omitted upon first submission. Abbreviations - NGPS - Next-Generation Protein Sequencing - LNPs - lipid nanoparticles - ROS - recognizer-ordered sequences - NAA - N-terminal amino acid - MAPE - mean absolute percent error - LOD - limit of detection - FDR - false discovery rate