Protein sequencing with single amino acid resolution discerns peptides that discriminate tropomyosin proteoforms

Abstract

Protein variants of the same gene--proteoforms--can have high molecular similarity yet exhibit different biological functions. Thus, identifying unique peptides that unambiguously map to proteoforms can provide crucial biological insights. In humans, four human tropomyosin (TPM) genes produce similar proteoforms that can be challenging to distinguish with standard proteomics tools. For example, TPM1 and TPM2 share 85% sequence identity, with amino acid substitutions that play unique roles in muscle contraction and myopathies. In this study, we evaluated the ability of the recently released Platinum single-molecule protein sequencer to detect proteoform-informative peptides. Platinum employs fluorophore-labeled recognizers that reversibly bind to cognate N-terminal amino acids (NAAs), enabling polypeptide sequencing within nanoscale apertures of a semiconductor chip that can accommodate single peptide molecules. As a proof of concept, we evaluated the ability of Platinum to distinguish three main types of proteoform variation: paralog-level, transcript-level, and post-translational modification (PTM). We distinguished paralogous TPM1 and TPM2 peptides differing by a single isobaric residue (leucine/isoleucine). We also distinguished tissue-specific TPM2 spliceforms. Notably, we found that a phosphotyrosine-modified peptide displayed reduced recognizer affinity for tyrosine, showing sensitivity to PTMs. This study paves the way for the targeted detection of proteoform biomarkers at the single molecule level. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/621980v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@122c6eaorg.highwire.dtl.DTLVardef@315f7forg.highwire.dtl.DTLVardef@1748af2org.highwire.dtl.DTLVardef@4e2b21_HPS_FORMAT_FIGEXP M_FIG C_FIG