Highly sensitive in situ proteomics with cleavable fluorescent tyramide reveals human neuronal heterogeneity

Liao, Renjie; Mondal, Manas; Nazaroff, Christopher D.; Mastroeni, Diego; Coleman, Paul D.; Guo, Jia

doi:10.1101/539106

Cited by 3 publications

(4 citation statements)

References 62 publications

(73 reference statements)

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“…We then quantified and compared the cleavage efficiency of 1-linker, 2linker, and 3-linker CFTz (Figure 4C). 1-linker CFTz has the cleavage efficiency of ~95.5%, which is consistent with our previously developed cleavable fluorescent probes (12,(17)(18)(19)(20). With multiple cleavage sites, 2-linker, and 3-linker CFTz increased the cleavage efficiency to ~97.5%.…”

Section: Enhanced Cleavage Efficiency By Cftz With Multiple Cleavage ...supporting

confidence: 88%

“…To enable single-cell spatial proteomic analysis, cyclic immunofluorescence and mass spectrometry imaging have been developed (6). Although these approaches (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) allow a large number of proteins to be quantified in their native cellular contexts with the subcellular resolution, some nonideal factors still exist. For instance, in several methods, antibodies are directly labeled with fluorophores or metal isotopes.…”

Section: Introductionmentioning

confidence: 99%

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Ultrasensitive and multiplexed protein imaging with clickable and cleavable fluorophores

Pham,

Chen,

Labaer

et al. 2023

Preprint

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Single-cell spatial proteomic analysis holds great promise to advance our understanding of the composition, organization, interaction and function of the various cell types in complex biological systems. However, the current multiplexed protein imaging technologies suffer from low detection sensitivity, limited multiplexing capacity or technically demanding. To tackle these issues, here we report the development of a highly sensitive and multiplexed in situ protein profiling method using off-the-shelf antibodies. In this approach, the protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and cleavable fluorophores via click chemistry. Through reiterative cycles of target staining, fluorescence imaging, and fluoropohore cleavage, many proteins can be profiled in single cells in situ. Applying this approach, we successfully quantified 28 different proteins in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue, which represents the highest multiplexing capacity among the tyramide signal amplification (TSA) methods. Based on their unique protein expression patterns and their microenvironment, ~820,000 cells in the tissue are classified into distinct cell clusters. We also explored the cell-cell interactions between these varied cell clusters and observed different subregions of the tissue are composed of cells from specific clusters.

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Section: Enhanced Cleavage Efficiency By Cftz With Multiple Cleavage ...supporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Ultrasensitive and multiplexed protein imaging with clickable and cleavable fluorophores

Pham,

Chen,

Labaer

et al. 2023

Preprint

Self Cite

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show abstract

“…To enable highly sensitive and multiplexed protein imaging with off-the-shelf antibodies, our group developed a reiterative protein staining approach using cleavable fluorescent tyramide (CFT) [ 17 ]. We demonstrated that its sensitivity is improved by about two orders of magnitude compared with other existing methods.…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, with tris(2-carboxyethyl) phosphine (TCEP) as the signal removal reagent, the first-generation CFT requires 65 °C to remove ~95% of the staining signals. Therefore, this relatively high reaction temperature could damage the integrity of the epitopes [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

et al. 2021

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Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.

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Deep Profiling of Cellular Heterogeneity by Emerging Single‐Cell Proteomic Technologies

2019

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The ability to comprehensively profile cellular heterogeneity in functional proteome is crucial in advancing the understanding of cell behavior, organism development, and disease mechanisms. Conventional bulk measurement by averaging the biological responses across a population often loses the information of cellular variations. Single‐cell proteomic technologies are becoming increasingly important to understand and discern cellular heterogeneity. The well‐established methods for single‐cell protein analysis based on flow cytometry and fluorescence microscopy are limited by the low multiplexing ability owing to the spectra overlap of fluorophores for labeling antibodies. Recent advances in mass spectrometry (MS), microchip, and reiterative staining‐based techniques for single‐cell proteomics have enabled the evaluation of cellular heterogeneity with high throughput, increased multiplexity, and improved sensitivity. In this review, the principles, developments, advantages, and limitations of these advanced technologies in analysis of single‐cell proteins, along with their biological applications to study cellular heterogeneity, are described. At last, the remaining challenges, possible strategies, and future opportunities that will facilitate the improvement and broad applications of single‐cell proteomic technologies in cell biology and medical research are discussed.

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Highly sensitive in situ proteomics with cleavable fluorescent tyramide reveals human neuronal heterogeneity

Cited by 3 publications

References 62 publications

Ultrasensitive and multiplexed protein imaging with clickable and cleavable fluorophores

Ultrasensitive and multiplexed protein imaging with clickable and cleavable fluorophores

Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

Deep Profiling of Cellular Heterogeneity by Emerging Single‐Cell Proteomic Technologies

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