Programmable Multiplexed Proteomics via Sequence-Encoded Mass Tagging

Isobaric tagging has revolutionized high-throughput proteomics by enabling multiplexed quantification, yet its reliance on isotopic encoding imposes fundamental limitations on scalability, synthesis complexity, and cost. Here, we introduce ePAS (expandable Platform by Arrangement of Sequence), a novel isotope-free tagging strategy that replaces isotopic elements with sequence-defined chemical modules. ePAS incorporates noncanonical amino acids (ncAAs) and a proline-based cleavage-assisting moiety to generate sequence-specific reporters upon MS/MS (MS²) fragmentation. This design enables a multiplexing platform whose theoretical capacity grows combinatorially with the number of ncAAs. As a proof-of-concept, we designed and synthesized a triplex ePAS tag set and demonstrated its robust performance in complex biological samples─including standard peptides, <i>Escherichia coli</i> (<i>E. coli</i>), and <i>Staphylococcus aureus</i> (<i>S. aureus</i>) lysates─achieving high labeling efficiency (>90%), accurate quantification (proportional error <20%), and broad dynamic range. Our work establishes ePAS as a universal, synthesis-friendly, and programmable platform that overcomes the intrinsic limitations of isotopic tags, opening new avenues for ultrahigh-throughput proteomics in clinical and single-cell applications.