Cargando…

Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidi...

Descripción completa

Detalles Bibliográficos
Autores principales: Liao, Renjie, Pham, Thai, Mastroeni, Diego, Coleman, Paul D., Labaer, Joshua, Guo, Jia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226835/
https://www.ncbi.nlm.nih.gov/pubmed/32244728
http://dx.doi.org/10.3390/cells9040852
_version_ 1783534373231919104
author Liao, Renjie
Pham, Thai
Mastroeni, Diego
Coleman, Paul D.
Labaer, Joshua
Guo, Jia
author_facet Liao, Renjie
Pham, Thai
Mastroeni, Diego
Coleman, Paul D.
Labaer, Joshua
Guo, Jia
author_sort Liao, Renjie
collection PubMed
description The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.
format Online
Article
Text
id pubmed-7226835
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-72268352020-05-18 Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin Liao, Renjie Pham, Thai Mastroeni, Diego Coleman, Paul D. Labaer, Joshua Guo, Jia Cells Article The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues. MDPI 2020-04-01 /pmc/articles/PMC7226835/ /pubmed/32244728 http://dx.doi.org/10.3390/cells9040852 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Liao, Renjie
Pham, Thai
Mastroeni, Diego
Coleman, Paul D.
Labaer, Joshua
Guo, Jia
Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin
title Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin
title_full Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin
title_fullStr Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin
title_full_unstemmed Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin
title_short Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin
title_sort highly sensitive and multiplexed in-situ protein profiling with cleavable fluorescent streptavidin
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226835/
https://www.ncbi.nlm.nih.gov/pubmed/32244728
http://dx.doi.org/10.3390/cells9040852
work_keys_str_mv AT liaorenjie highlysensitiveandmultiplexedinsituproteinprofilingwithcleavablefluorescentstreptavidin
AT phamthai highlysensitiveandmultiplexedinsituproteinprofilingwithcleavablefluorescentstreptavidin
AT mastroenidiego highlysensitiveandmultiplexedinsituproteinprofilingwithcleavablefluorescentstreptavidin
AT colemanpauld highlysensitiveandmultiplexedinsituproteinprofilingwithcleavablefluorescentstreptavidin
AT labaerjoshua highlysensitiveandmultiplexedinsituproteinprofilingwithcleavablefluorescentstreptavidin
AT guojia highlysensitiveandmultiplexedinsituproteinprofilingwithcleavablefluorescentstreptavidin