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In vivo mRNA display enables large-scale proteomics by next generation sequencing
- Source :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Year :
- 2020
- Publisher :
- National Academy of Sciences, 2020.
-
Abstract
- Significance The need for mass-spectrometric determination of protein identity severely limits the throughput of many whole-cell proteomic analyses. We have developed an approach to physically connect in vivo-expressed proteins to their encoding messenger RNAs (mRNAs). This genotype–phenotype linkage enables us to carry out a variety of whole-proteome assays and determine the identity of each protein using nucleic acid sequencing. We show that the resulting in vivo mRNA display technology enables determination of protein subcellular localization and protein–protein interactions, with high sensitivity and specificity, using simple purification followed by high-throughput sequencing. In vivo mRNA display should enable more efficient interrogation of proteome behavior in a variety of applications that are currently limited by the cost and throughput of mass-spectrometric analysis.<br />Large-scale proteomic methods are essential for the functional characterization of proteins in their native cellular context. However, proteomics has lagged far behind genomic approaches in scalability, standardization, and cost. Here, we introduce in vivo mRNA display, a technology that converts a variety of proteomics applications into a DNA sequencing problem. In vivo-expressed proteins are coupled with their encoding messenger RNAs (mRNAs) via a high-affinity stem-loop RNA binding domain interaction, enabling high-throughput identification of proteins with high sensitivity and specificity by next generation DNA sequencing. We have generated a high-coverage in vivo mRNA display library of the Saccharomyces cerevisiae proteome and demonstrated its potential for characterizing subcellular localization and interactions of proteins expressed in their native cellular context. In vivo mRNA display libraries promise to circumvent the limitations of mass spectrometry-based proteomics and leverage the exponentially improving cost and throughput of DNA sequencing to systematically characterize native functional proteomes.
- Subjects :
- Proteomics
Saccharomyces cerevisiae Proteins
Proteome
Context (language use)
Computational biology
protein–protein interactions
Biology
DNA sequencing
Protein–protein interaction
Protein Interaction Mapping
mRNA display
RNA, Messenger
DNA, Fungal
protein display technologies
Gene Library
Multidisciplinary
Base Sequence
RNA
High-Throughput Nucleotide Sequencing
Sequence Analysis, DNA
Biological Sciences
Applied Biological Sciences
Binding domain
MS2 tagging
Subjects
Details
- Language :
- English
- ISSN :
- 10916490 and 00278424
- Volume :
- 117
- Issue :
- 43
- Database :
- OpenAIRE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Accession number :
- edsair.doi.dedup.....9364d218902a9b6ebf8b6fb8e17b5ca2