Coupling detection of distinct RNA isoforms and alternatively spliced proteoforms is a key goal in proteogenomics. For example, actin-binding tropomyosin (TPM1-4) genes generate over 40 spliceoforms in mammals, several of which are functionally distinct. Notably, genetic and transcriptomic studies in human models have associated several TPM spliceoforms with complex diseases such as osteoporosis and coronary artery disease. However, subtle amino acid variations hamper unequivocal identification of TPM proteoforms. Here we integrate LR-RNA seq (PacBio) of transcript isoforms with Platinum (Quantum-Si), a benchtop platform that reveals the primary structure of single protein molecules, to distinguish TPM1/2 (87% amino acid identity).

We created a database for long read-guided interpretation of protein isoforms and constructed a map of protein isoforms for splicing events associated with bone mineral density in the human fetal osteoblast (hFOB) cell line. We derived a list of TPM1/2-informative peptides and TPM2 isoform-informative peptides from our long-read transcript database, then utilized Platinum to differentiate synthetic peptides that correspond to TPM1/2 paralogs and spliceoforms.

In the Platinum workflow, derivatized peptides are conjugated via C-termini and loaded into nanoscale reaction chambers of a semiconductor chip, with the goal of one peptide per aperture. During sequencing, dye-labeled N-terminal amino acid (NAA) recognizers reversibly bind to cognate NAAs, generating distinct binding and pulsing patterns. Aminopeptidases sequentially cleave NAAs to expose subsequent amino acids for recognition. Upon completion of a sequencing run, data is transmitted for cloud-based analysis. Traces are segmented into contiguous groups of pulses, or recognition segments (RSs), each representing interactions between recognizers and NAAs. The order of recognizer binding and kinetic properties of RSs are aligned to reference sequences to determine peptide sequence. Platinum sequencing discriminates TPM peptides with similar physicochemical properties.

NAA recognizers that bind glutamate (E) distinguish SLEAQAEK (TPM1) and SLEAQADK (TPM2). Also, differential pulse durations (PDs) discern isobaric TPM1/2 peptides. At the exon level, NAA recognizers for aromatic residues reveal an isoform-informative peptide in TPM2. Overall, single molecule sequencing with orthogonal methods facilitates proteogenomics and the transition from the discovery to proteoform scoring phase.