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

Figures

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  1. Sequence alignment of TPM1 and TPM2 isoforms and relevance of amino acid mutations to pathology. The primary structures of canonical TPM1 and two TPM2 spliceforms are shown. TPM and troponin associate with actin to form the contractile apparatus. Region A corresponds to an N-terminal region that contains isobaric peptides that differ by leucine (Leu; L) or isoleucine (Ile; I). Mutations in these paralogues have been linked with hypertrophic cardiomyopathy (HCM). Region B delineates an internal eight amino acid long region within TPM1/2 paralogues. Mutations within this region are also found in HCM and joint deformities due to the Sheldon-Hall syndrome and distal arthrogryposis. − Region C highlights an isoform-informative peptide (SLMASEEEYSTK) that can be used as a cancer marker. , Region D maps to an alternatively spliced C-terminal region of TPM2-201 and TPM2-210. While TIDDLEETLASAK (TPM2-201) and TIDDLEDEVYAQK (TPM2-210) share the same amino acid length, TIDDLEDEVYAQK is subject to phosphorylation on a single tyrosine residue. ,

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  2. Overview of sequencing on Platinum. (A) The Quantum-Si NGPS platform consists of a semiconductor chip, sample prep and sequencing kits, the compact Platinum instrument (27 lbs), and cloud-based software analysis. (B) The semiconductor chip contains discrete nanoapertures spread across two flow cells that can accommodate single peptide molecules. The sequencing kits employ aminopeptidases and six N-terminal amino acid (NAA) recognizers, which are labeled with different fluorophores. (C) During sequencing, recognizers reversibly bind NAAs, generating characteristic pulsing regions called recognition segments (RSs). As NAA recognizers can target 1–3 NAAs, distinct pulse duration (PD) provides a kinetic readout of NAA identity. (D) Upon completion of a sequencing run, data is automatically transmitted for cloud-based analysis. Reads are then aligned to the reference profile based on the correspondence of observed RSs to the expected reference profile, using recognizer identity. Kinetic signature numerical values (RS start, PD) represent the median of a distribution of data across analyzed apertures that contains 4 RS and 3 unique dyes.

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  3. Kinetic signatures discern the identity and amino acid ordering of paralogue-derived peptides. (A) Gene duplication events produce TPM1/TPM2 paralogues, wherein a single isobaric Leu to Ile substitution differentiates ENALDRAEQAEADK from ENAIDRAEQAEADK (top). An example kinetic signature snapshot from cloud-based analysis is shown below the amino acid positions (bottom). Colors of the boxes indicate the recognizer identity (see Figure B). Kinetic signatures such as PD represent the statistical distribution of kinetic data for all pulses associated with a specific residue. (B) Plot of the fold change in the average pulse duration (PD) between NAA measurements of ENAL versus ENAI, for the first 4 residues. Error bars indicate standard deviation from three experiments conducted on independent flow cells. Asterisks indicate p-values relative to a PD fold change of 1. (C) Gene duplication events produce TPM1/TPM2 paralogues that can be differentiated at position 4 of TPM1 peptide VIESRAQK and TPM2 peptide VIENRAMK. (D) Plot of the fold change PDs for VIENSRAQ versus VIENRAM. A ∼5-fold higher PD was detected for the Asn in position 4 of VIENRAMK, relative to Ser in VIESRAQK, which was…

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  4. Kinetic signatures discern the identity and temporal order of TPM2 peptides derived from alternative splicing. (A) Alternative splicing of TPM2 mRNA generates a tissue-specific spliceform (containing Exon 6) to which SLMASEEEYSTK specifically maps. (B) Sequence alignments for SLMASEEEYSTK. An example kinetic signature snapshot from cloud-based analysis is shown below the amino acid positions. Colors of the boxes indicate the recognizer identity (see Figure B). Kinetic signatures such as PD represent the statistical distribution of kinetic data for all pulses associated with a specific residue. (C) Alternative splicing of TPM2 mRNA differentiates exon-specific peptides TIDDLEETLASAK (Exon 10) and TIDDLEDEVYAQK (Exon 11). (D) Sequence alignments for peptides TIDDLEETLASAK and TIDDLEDEVYAQK. (E) LIV recognizer elicits differential PD profiles for similar aliphatic residues. A ∼9-fold difference in PD was observed for Leu/Val. (F) Distinct recognizers for Ala and Tyr discern position 9. (G) The Ala/Ser recognizer enables discernment via differential PDs at position 10. Data (*mean ± S.D.) are representative of three independent experiments, conducted on independent flow cells (ns p >…

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  5. YFW recognizer generates Y-specific recognition events, enabling differentiation from pY-modified peptidoforms. (A) Schema showing the exon-specific peptide TIDDLEDEVYAQK, which contains a known phosphosite at the Tyr residue. (B) Bar plot showing the difference in percent of apertures between the recognizers (LIV, E, and YFW) in the unmodified versus phosphorylated version (TIDDLEDEVpYAQK). Data (mean ± S.D.) are representative of three independent experiments, conducted on independent flow cells (ns p > 0.05, ***p < 0.001; Welch two-sample t-test).