Cysteine(Cys)-specific bioconjugation has widespread applications in the synthesis of protein conjugates,particularly for the functionalization of antibodies.Here,we report the discovery of transstyryl sulfonyl fluori...Cysteine(Cys)-specific bioconjugation has widespread applications in the synthesis of protein conjugates,particularly for the functionalization of antibodies.Here,we report the discovery of transstyryl sulfonyl fluoride(SSF)as a near-perfect Michael acceptor for Cys-specific protein bioconjugation.Compared to maleimides,which are predominantly used,SSF exhibited better chemoselectivity,selfstability,and conjugate stability while maintaining comparable reactivity.Using SSF-derived probes,proteins can be readily modified on the Cys residue(s)to install functionalities,for example,fluorescent dyes,toxins,and oligonucleotides,without influencing the activity.Further applications of SSF-derived serum-stable antibody-drug conjugates and PD-L1 nanobody-oligo conjugates demonstrate the great translational value of SSF-based bioconjugation in drug development and single-cell sequencing.展开更多
Cellular indexing of transcriptomes and epitopes by sequencing(CITE-seq)enables the simultaneous analysis of transcriptomic and proteomic data at the single-cell level,providing a comprehensive view of cellular hetero...Cellular indexing of transcriptomes and epitopes by sequencing(CITE-seq)enables the simultaneous analysis of transcriptomic and proteomic data at the single-cell level,providing a comprehensive view of cellular heterogeneity and function.In this study,we present a standardized approach for high-quality single-cell RNA sequencing coupled with cell surface protein quantification.Key advantages of CITE-seq include its compatibility with existing scRNA-seq workflows,cost-efficient high-throughput protein detection,and enhanced resolution in cell type classification.Detailed steps for sample preparation,antibody-oligo conjugation,gel bead-in-emulsion(GEM)generation,complementary deoxyribonucleic acid(cDNA)amplification,and library construction are provided,ensuring reproducibility and robust data quality.This protocol facilitates the integration of multimodal single-cell data,enabling precise characterization of rare cell subsets and advancing insights in immunology,oncology,and developmental biology.The workflow is optimized for flexibility across platforms and scalable for diverse research applications.展开更多
基金Financial support from the National Key R&D Program of China(grant no.2019YFA09006600)the National Natural Science Foundation of China(grant nos.21977048 and 92053111)+2 种基金the Natural Science Foundation of Jiangsu Province(grant no.BK20202004)the Beijing National Laboratory for Molecular Sciences(grant no.BNLMS20200)the Jiangsu Specially-Appointed Professor Plan,and the Program for Innovative Talents and Entrepreneur in Jiangsu is gratefully acknowledged.Q.Z.is the Connie and Bob Lurie Fellow of the Damon Runyon Cancer Research Foundation(DRG-2434-21).
文摘Cysteine(Cys)-specific bioconjugation has widespread applications in the synthesis of protein conjugates,particularly for the functionalization of antibodies.Here,we report the discovery of transstyryl sulfonyl fluoride(SSF)as a near-perfect Michael acceptor for Cys-specific protein bioconjugation.Compared to maleimides,which are predominantly used,SSF exhibited better chemoselectivity,selfstability,and conjugate stability while maintaining comparable reactivity.Using SSF-derived probes,proteins can be readily modified on the Cys residue(s)to install functionalities,for example,fluorescent dyes,toxins,and oligonucleotides,without influencing the activity.Further applications of SSF-derived serum-stable antibody-drug conjugates and PD-L1 nanobody-oligo conjugates demonstrate the great translational value of SSF-based bioconjugation in drug development and single-cell sequencing.
基金supported by the Chinese Academy of Medical Sciences(CAMS)Innovation Funds for Medical Sciences(2024-I2M-3-001).
文摘Cellular indexing of transcriptomes and epitopes by sequencing(CITE-seq)enables the simultaneous analysis of transcriptomic and proteomic data at the single-cell level,providing a comprehensive view of cellular heterogeneity and function.In this study,we present a standardized approach for high-quality single-cell RNA sequencing coupled with cell surface protein quantification.Key advantages of CITE-seq include its compatibility with existing scRNA-seq workflows,cost-efficient high-throughput protein detection,and enhanced resolution in cell type classification.Detailed steps for sample preparation,antibody-oligo conjugation,gel bead-in-emulsion(GEM)generation,complementary deoxyribonucleic acid(cDNA)amplification,and library construction are provided,ensuring reproducibility and robust data quality.This protocol facilitates the integration of multimodal single-cell data,enabling precise characterization of rare cell subsets and advancing insights in immunology,oncology,and developmental biology.The workflow is optimized for flexibility across platforms and scalable for diverse research applications.
