Protein Sequencing with Single Amino Acid Resolution Discerns Peptides That Discriminate Tropomyosin Proteoforms

Area of Science:

• Proteomics
• Molecular Biology
• Biotechnology

Background:

• Protein variants, or proteoforms, share high molecular similarity but have distinct biological functions.
• Distinguishing between similar proteoforms is challenging for standard proteomics techniques.
• Human tropomyosin (TPM) genes exemplify this challenge, with TPM1 and TPM2 sharing 85% sequence identity.

Purpose of the Study:

• To evaluate the Platinum single-molecule protein sequencer's capability in identifying proteoform-informative peptides.
• To demonstrate the sequencer's ability to differentiate between paralogue, transcript, and post-translational modification (PTM) levels of proteoform variation.

Main Methods:

• Utilized the Platinum sequencer, which employs fluorophore-labeled recognizers for N-terminal amino acid sequencing.
• Sequenced single peptide molecules within nanoscale apertures on a semiconductor chip.
• Applied the technology to distinguish between TPM1 and TPM2 paralogues, TPM2 spliceforms, and a phosphotyrosine-modified peptide.

Main Results:

• Successfully distinguished between paralogous TPM1 and TPM2 peptides with a single leucine/isoleucine difference.
• Differentiated tissue-specific spliceforms of TPM2.
• Observed reduced recognizer affinity for a phosphotyrosine-modified peptide, indicating PTM sensitivity.

Conclusions:

• The Platinum single-molecule protein sequencer can effectively identify unique peptides for distinguishing complex proteoforms.
• This technology shows promise for targeted detection of proteoform biomarkers at the single-molecule level.
• Advances in single-molecule sequencing open new avenues for understanding protein diversity and function.