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.

LC-MS/MS analysis Peptide separation was performed using nanoflow high-performance liquid chromatography (HPLC) on a Dionex Ultimate 3000 system (Thermo Fisher Scientific, Bremen, Germany). Peptides were initially loaded onto an Acclaim PepMap 100 trap column (300 μm × 5 mm, 5 μm C18), followed by gradient elution through an Acclaim PepMap 100 analytical column (75 μm × 25 cm, 3 μm C18) for enhanced separation. Mass spectrometry (MS) analysis was conducted using an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with the Orbitrap Eclipse Tune (version 4.0.4091) and Xcalibur software (version 4.5.445.18) for data acquisition and analysis.

Sup Fig 2 Peptide File name Sample ENAL/IDRAEQAEDK 240502_AC12_NS_16peptides_DDA_49min.raw Synthetic peptide mixture VIENRAMK 240502_AC12_NS_16peptides_DDA_49min.raw Synthetic peptide mixture VIESRAQK 240502_AC12_NS_16peptides_DDA_49min.raw Synthetic peptide mixture SLMASEEEYSTK 240502_AC12_NS_16peptides_DDA_49min.raw Synthetic peptide mixture TIDDLEETLASAK 240502_AC12_NS_16peptides_DDA_49min.raw Synthetic peptide mixture TIDDLEDEVYAQK 240502_AC12_NS_16peptides_DDA_49min.raw Synthetic peptide mixture Sup Fig 4 Peptide File name Sample VIENRAMK 240502_AC12_NS_TPM2_DDA_49min.raw Recombinant TPM2 LysC digest ENALDRAEQAEADK 240502_AC12_NS_TPM1_plus_2_DDA_49min.raw Recombinant TPM1andTPM2 LysC digest ENAIDRAEQAEADK 240502_AC12_NS_TPM1_plus_2_DDA_49min.raw Recombinant TPM1andTPM2 LysC digest