MSACL 2025 Abstract

Exploratory Application of Semiconductor Chip-Based Single-Molecule Protein Sequencing for Confirmation of Hemoglobin Variants

Ruben Y. Luo (1,2), Mathivanan Chinnaraj (3), Carolyn V. Wong (2), Jianan Lin (3), Kristin Blacklock (3), Douglas Pike (3), Ilya Chorny (3), John Vieceli (3), Priscilla S.-W. Yeung (1,2)
(1) Department of Pathology, School of Medicine, Stanford University, Stanford, CA, USA (2) Clinical Laboratories, Stanford Health Care, Palo Alto, CA, USA (3) Quantum-Si, Branford, CT, USA

Ruben Y. Luo, PhD, DABCC (Presenter) Stanford University

Presenter Bio: Ruben Y. Luo, PhD, DABCC, FADLM is an Assistant Professor of Pathology at Stanford University and an Associate Director of Clinical Chemistry Laboratory at Stanford Health Care. He received PhD in chemistry from Stanford University, worked in the clinical diagnostic industry for several years, and then completed clinical chemistry fellowship at the University of California San Francisco. Dr. Luo is dedicated to innovations in clinical diagnostics. His research focuses on (1) discovering the clinical diagnostic value of molecular characteristics of protein biomarkers, and (2) applying top-down mass spectrometry and label-free optical sensing immunoassays to characterization and accurate measurement of biomarkers. He has been an active member and conference speaker in the international clinical chemistry and mass spectrometry communities, e.g., Association for Diagnostics and Laboratory Medicine (ADLM; formerly American Association for Clinical Chemistry, AACC), American Society for Mass Spectrometry (ASMS), Mass Spectrometry & Advances in Clinical Lab (MSACL). His research awards include 2022 AACC George Grannis Award for Excellence in Research and Scientific Publication, 2020 American Society for Clinical Pathology (ASCP) “40 Under Forty” Honoree, etc. He currently serves as an associate editor of JMSACL and an editorial board member of Scientific Reports.

Relevant Financial Disclosures (within past 24 months, reported on Apr 21, 2026) Other Potential Conflicts Thermo Fisher Scientific (San Jose, CA) / Research Collaboration Gator Bio (Palo Alto, CA) / 2 Seed Instruments Instant Nanobiosciences (New Taipei City, Taiwan) / 1 Seed Instrument CMP Scientific (Brooklyn, NY) / 1 Seed Instrument Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Proteins are a major group of clinical diagnostic markers. Many clinical tests for protein diagnostic markers are primarily based on superficial molecular features of proteins such as specific reactivity for enzymatic assays or epitope binding for immunoassays. Besides quantifying proteins, these technologies provide little information about primary structural characteristics. Advanced technologies, such as mass spectrometry (MS), together with proteomic methodologies are being introduced to the field of clinical diagnostics to facilitate the characterization of protein structures. Among these, the newly emerged single-molecule protein sequencing (SMPS) technologies are promising because they provide the unique ability to observe the complete primary structure of every single protein molecule instead of measuring an ensemble average of numerous molecules.(1) This advantage can facilitate ultimate analytical sensitivity and unprecedented resolution in measuring proteins of interest. Currently two representative technologies of this type are semiconductor chip-based and nanopore-based SMPS.(2,3)

Identification of hemoglobin (Hb) variants is of significant value in the clinical diagnosis of hemoglobinopathy. As the major respiratory protein in red blood cells, Hb is a heterotetramer consisting of two pairs of subunits. The major Hb subunits include α, β, γ, and δ, which consitute HbA (α2β2), HbA2 (α2δ2), HbF (α2γ2), etc. About 1400 amino acid variants of Hb subunits in human have been reported, mostly those of α and β. The conventional methods to identify Hb variants in clinical laboratories can mostly narrow down the range of candidates for a Hb variant sample, but are not able to pinpoint the exact Hb variant. A semiconductor chip-based SMPS approach was explored for identification of Hb variants.

METHODS:
A semiconductor chip-based SMPS method using a Quantum-Si sequencer (Quantum- Si, Branford, CT) was applied to confirm Hb variants in clinical samples using the technology. A Hb sample was denatured and digested to peptides, which were then derived to connect to a linker. The individual peptide molecules are distributed into attoliter-sized reaction chambers on a semiconductor chip and immobilized on the chip surface. Recognizers, i.e., proteins derived from bacteria or yeast that bind to specific amino acid residues, are added to the reaction chambers to bind the N-terminal amino acids (NAAs) of the peptides. Pulsed laser light is routed to illuminate each reaction chamber and excite the different fluorophores linked to the recognizers. With the presence of aminopeptidases in each reaction chamber, the NAA of the peptide molecule is removed after a certain period of time and the second-to-the-last amino acid becomes the new NAA, allowing for the next cycle of recognizer binding. The sequential cycles of NAA-recognizer binding facilitate the reading of the peptide amino acid sequence, and the sequenced peptides can be matched to a protein database to identify the original proteins in the sample with minimum of 4 recognition segments needed of 3 recognizers to result in an alignment.(4)

RESULTS:
Two Hb variant samples were analyzed by SMPS, which had been tested with conventional electrophoretic and more advanced mass spectrometric methods. The SMPS analysis detected the mutated proteotypic peptides of the correct Hb variants: α/α-Handsworth (G18R) and β/β-Accra (D73N). To confirm the SMPS results, synthetic proteotypic peptides corresponding to the two Hb variant samples were also analyzed.

CONCLUSION:
We have performed the semiconductor chip-based SMPS for structural characterization of hemoglobin variant subunits. With the use of recognizers for N-terminal amino acids and aminopeptidases, the proteotypic peptides of the variant subunits could be detected and identified. This exploratory application of SMPS supports the potential of future use of SMPS technologies in clinical laboratories.

REFERENCES:
1. Yeung PSW, Luo RY. Transport of Full-Length Proteins through a Nanopore: One Step Closer to Single-Molecule Proteomics. Clinical Chemistry. 2024;70:462–3.
2. Reed BD, Meyer MJ, Abramzon V, Ad O, Ad O, Adcock P, et al. Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device. Science. 2022;378:186–92.
3. Wei X, Penkauskas T, Reiner JE, Kennard C, Uline MJ, Wang Q, et al. Engineering Biological Nanopore Approaches toward Protein Sequencing. ACS Nano. 2023;17:16369–95.
4. Chinnaraj M, Lin J, Blacklock K, Hermes E, Meyer M, Pike D, et al. Detecting Amino Acid Variants Using Next-Generation Protein Sequencing (NGPS) [Internet]. 2024 [cited 2025 Mar 19]. Available from: http://biorxiv.org/lookup/doi/10.1101/2024.12.17.629036.