Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device

Proteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

Protein Sequencing, One Molecule at a Time

Despite tremendous gains over the past decade, methods for characterizing proteins have generally lagged behind those for nucleic acids, which are characterized by extremely high sensitivity, dynamic range, and throughput. However, the ability to directly characterize proteins at nucleic acid levels would address critical biological challenges such as more sensitive medical diagnostics, deeper protein quantification, large-scale measurement, and discovery of alternate protein isoforms and modifications and would open new paths to single-cell proteomics. In response to this need, there has been a push to radically improve protein sequencing technologies by taking inspiration from high-throughput nucleic acid sequencing, with a particular focus on developing practical methods for single-molecule protein sequencing (SMPS). SMPS technologies fall generally into three categories: sequencing by degradation (e.g., mass spectrometry or fluorosequencing), sequencing by transit (e.g., nanopores or quantum tunneling), and sequencing by affinity (as in DNA hybridization–based approaches). We describe these diverse approaches, which range from those that are already experimentally well-supported to the merely speculative, in this nascent field striving to reformulate proteomics. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone receptor 1

Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to Gs and increased activation of Gq/11 compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity.

Resolving isomeric posttranslational modifications using a nanopore

Posttranslational modifications (PTMs) of proteins are crucial for cellular function but pose analytical problems, especially in distinguishing chemically identical PTMs at different nearby locations within the same protein. Current methods, such as liquid chromatography-tandem mass spectrometry, are technically tantamount to de novo protein sequencing. Nanopore techniques may provide a more efficient solution, but applying the concepts of nanopore DNA strand sequencing to proteins still faces fundamental problems. Here, we demonstrate the use of an engineered biological nanopore to differentiate positional isomers resulting from acetylation or methylation of histone protein H4, an important PTM target. In contrast to strand sequencing, we differentiate positional isomers by recording ionic current modulations resulting from the stochastic entrapment of entire peptides in the pore's sensing zone, with all residues simultaneously contributing to the electrical signal. Molecular dynamics simulations show that, in this whole-molecule sensing mode, the non-uniform distribution of the electric potential within the nanopore makes the added resistance contributed by a PTM dependent on its precise location on the peptide. Optimization of the pore's sensitivity in combination with parallel recording and automated and standardized protein fragmentation may thus provide a simple, label-free, high-throughput analytical platform for identification and quantification of PTMs.

Planar Boronic Graphene and Nitrogenized Graphene Heterostructure for Protein Stretch and Confinement

Single-molecule techniques such as electron tunneling and atomic force microscopy have attracted growing interests in protein sequencing. For these methods, it is critical to refine and stabilize the protein sample to a “suitable mode” before applying a high-fidelity measurement. Here, we show that a planar heterostructure comprising boronic graphene (BC3) and nitrogenized graphene (C3N) sandwiched stripe (BC3/C3N/BC3) is capable of the effective stretching and confinement of three types of intrinsically disordered proteins (IDPs), including amyloid-β (1–42), polyglutamine (Q42), and α-Synuclein (61–95). Our molecular dynamics simulations demonstrate that the protein molecules interact more strongly with the C3N stripe than the BC3 one, which leads to their capture, elongation, and confinement along the center C3N stripe of the heterostructure. The conformational fluctuations of IDPs are substantially reduced after being stretched. This design may serve as a platform for single-molecule protein analysis with reduced thermal noise.

M-Protein Sequencing and Monitoring in Serum of LC-Only Multiple Myeloma Patients

Background M-protein, a secreted antibody of malignant plasma cells, is a gold standard biomarker for monitoring the disease status in multiple myeloma (MM) patients (pts). The development of peripheral blood based ultrasensitive (MRD) methods of M protein detection is of high interest and importance. In 20% of MM pts, the M-protein consists of only the light chain (LC) of the immunoglobulin (Ig) molecule. For these LC-only MM pts, disease monitoring is challenging due to low levels of M-protein in serum. Urine protein electrophoresis (UPEP) and immunofixation electrophoresis (IFE), or serum free light chain (FLC) lack the sensitivity and/or specificity to track the M-protein in these pts, while bone marrow-based assays cannot be performed frequently due to their invasive nature. Thus, we evaluated the performance of EasyM, a mass spectrometry(MS)-based, non-invasive, sensitive assay for monitoring M-protein levels in LC-only patients in the MCRN-001 Canadian national and MD Anderson VRD-panobinostat frontline trials. Methods MCRN-001 trial is evaluating enhanced conditioning prior to ASCT for newly diagnosed MM (NDMM). After treatment with bortezomib (BTZ) based induction eligible MM pts received BuMel prior to ASCT. Busulfan was administered via IV at 3.2 mg/kg on days -5 to -3, or days -6 to -4 pre-ASCT (day 0) and melphalan was given at 140 mg/m 2 on day -2 or -3 pre-ASCT. Lenalidomide (LEN) administration began 100 days post-ASCT at 10 mg/d and continued until progressive disease (PD) onset. In the MDACC 2011-0192 frontline study in newly diagnosed MM, transplant-eligible pts received the novel combination of LEN, BTZ, dexamethasone (DEX) and panobinostat (RVD-panobinostat). The IMWG criteria were used to monitor clinical response in both trials. A total of 13 LC-only MM pts were selected for the study. Local IRB approval was obtained prior to the study. To derive the M-protein's full amino acid sequence, FLC was first enriched from the diagnostic serum sample. The FLC enrichment consisted of IgG depletion with protein A/G beads, followed by affinity purification of kappa or lambda LC containing Igs. Non-reducing PAGE was then used to separate FLC monomers, FLC dimers and full-length Igs. Finally, in-gel digestion of FLC monomers and dimers by multiple proteolytic enzymes were analyzed on a Q-Exactive mass spectrometer. Data analysis and sequence assembly were performed with the REmAb protein sequencing platform. To monitor M-protein levels in serum, unique, tryptic peptides from sequenced FLC were selected and quantified in diagnostic and follow-up samples with a PRM assay on a Q-Exactive instrument. Results M-protein sequencing The full FLC sequence was derived for 8 out of 13 (61.5%) LC-only pts. The M-protein was successfully sequenced even when the FLC concentration was as low as 147 mg/L. However, FLC concentration measured by FreeLite assay was not a reliable predictor of our ability to derive the full sequence. The appearance of a sharp FLC monomer and dimer bands on PAGE after FLC enrichment was a better predictor of the M-protein sequencing success. M-protein monitoring The M-protein of one pt did not contain any unique peptides. This pt was excluded for further analysis. In the remaining 7 pts, the M-protein contained at least one unique peptide and could thus be monitored by EasyM. For 6 LC-only pts, the M-protein monitored by EasyM correlated with the disease status measured by serum FLC, UPEP and urine IFE. A separate serial dilution test estimated that the limit of quantification can reach as low as 0.13 mg/L. Figure 1 shows representative data for two LC-only patients. One pt experienced relapse during the study; however, this relapse could not be detected by EasyM. This result could indicate a possible clonal switch at time of disease progression, but further investigation is needed to verify this. Conclusions Due to the rapid turnover and clearance of light chains conventional blood/urine tests have lacked the higher sensitivity needed to monitor disease state in LC only MM patients. To overcome the lower FLC concentration the current study successfully applied LC enrichment strategies to enable the sequencing and high sensitivity monitoring of FLC M-protein by MS in blood. The EasyM LC assay is non-invasive, sensitive, capable of assessing disease status, and has potential to be further investigated as a peripheral blood myeloma response and MRD monitoring biomarker. Figure 1 Figure 1. Disclosures Ma: Rapid Novor Inc.: Current holder of individual stocks in a privately-held company. Reece: Millennium: Research Funding; Amgen: Consultancy, Honoraria; GSK: Honoraria; Karyopharm: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Honoraria; BMS: Honoraria, Research Funding. Manasanch: GSK, Secura Bio,Takeda, Celgene, Sanofi, Janssen and Adaptive Biotechnologies: Consultancy; Sanofi, Quest Diagnostics, Novartis, JW Pharma, Merck: Research Funding. Orlowski: Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, EcoR1 Capital LLC, Genzyme, GSK Biologicals, Janssen Biotech, Karyopharm Therapeutics, Inc., Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, Inc., Sanofi-Aventis, and Takeda P: Consultancy, Honoraria; CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Other: Clinical research funding; Asylia Therapeutics, Inc., BioTheryX, Inc., and Heidelberg Pharma, AG.: Other: Laboratory research funding; Asylia Therapeutics, Inc.: Current holder of individual stocks in a privately-held company, Patents & Royalties; Amgen, Inc., BioTheryX, Inc., Bristol-Myers Squibb, Celgene, Forma Therapeutics, Genzyme, GSK Biologicals, Janssen Biotech, Juno Therapeutics, Karyopharm Therapeutics, Inc., Kite Pharma, Neoleukin Corporation, Oncopeptides AB, Regeneron Pharmaceuticals, I: Membership on an entity's Board of Directors or advisory committees. Trudel: GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Roche: Consultancy; Genentech: Research Funding; Pfizer: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Sanofi: Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding.