Programmable Multiplexed Proteomics via Sequence-Encoded Mass Tagging

Area of Science:

• Proteomics
• Analytical Chemistry
• Biotechnology

Background:

• Isobaric tagging revolutionized proteomics quantification but faces limitations in scalability, synthesis complexity, and cost due to isotopic encoding.
• Existing methods hinder ultrahigh-throughput applications in clinical and single-cell research.

Purpose of the Study:

• Introduce ePAS (expandable Platform by Arrangement of Sequence), a novel isotope-free tagging strategy for proteomics.
• Overcome the intrinsic limitations of isotopic tags for enhanced scalability, synthesis, and cost-effectiveness.

Main Methods:

• Developed ePAS, utilizing sequence-defined chemical modules and noncanonical amino acids (ncAAs) instead of isotopic elements.
• Incorporated a proline-based moiety for sequence-specific reporter generation upon MS/MS fragmentation.
• Designed and synthesized a triplex ePAS tag set for proof-of-concept studies.

Main Results:

• Demonstrated robust performance in complex biological samples (peptides, E. coli, S. aureus lysates).
• Achieved high labeling efficiency (>90%), accurate quantification (proportional error <20%), and broad dynamic range.
• Showcased combinatorial growth in multiplexing capacity with increasing ncAAs.

Conclusions:

• ePAS is a universal, synthesis-friendly, and programmable platform overcoming isotopic tag limitations.
• This novel strategy enables ultrahigh-throughput proteomics.
• Opens new avenues for clinical and single-cell proteomics applications.