Discovery of protein interactions using parallel analysis of translated ORFs (PLATO)

Harry Larman, Anthony C. Liang, Stephen J. Elledge, Jian Zhu

Research output: Contribution to journalArticle

Abstract

Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA molecules to the proteins they encode, and recovered mRNA from affinity enrichment is subjected to analysis using massively parallel DNA sequencing. Compared with alternative in vitro methods, PLATO provides several advantages including library size and cost. A unique advantage of PLATO is that an alternative reverse transcription-quantitative PCR (RT-qPCR) protocol can be used to test binding of specific, individual proteins. To illustrate a typical experimental workflow, we demonstrate PLATO for the identification of the immune target of serum antibodies from patients with inclusion body myositis (IBM). Beginning with an ORFeome library in an RD vector, the protocol can produce samples for deep sequencing or RT-qPCR within 4 d.

Original languageEnglish (US)
Pages (from-to)90-103
Number of pages14
JournalNature Protocols
Volume9
Issue number1
DOIs
StatePublished - Jan 2014
Externally publishedYes

Fingerprint

Open Reading Frames
Transcription
Display devices
Immobilized Antibodies
Messenger RNA
Proteins
Libraries
High-Throughput Nucleotide Sequencing
Molecular weight
Ribosomes
Reverse Transcription
Peptides
Molecules
Antibodies
DNA
Inclusion Body Myositis
Polymerase Chain Reaction
Workflow
Costs
DNA Sequence Analysis

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Discovery of protein interactions using parallel analysis of translated ORFs (PLATO). / Larman, Harry; Liang, Anthony C.; Elledge, Stephen J.; Zhu, Jian.

In: Nature Protocols, Vol. 9, No. 1, 01.2014, p. 90-103.

Research output: Contribution to journalArticle

Larman, Harry ; Liang, Anthony C. ; Elledge, Stephen J. ; Zhu, Jian. / Discovery of protein interactions using parallel analysis of translated ORFs (PLATO). In: Nature Protocols. 2014 ; Vol. 9, No. 1. pp. 90-103.
@article{624dbb628663457bb08b5d437222c4ad,
title = "Discovery of protein interactions using parallel analysis of translated ORFs (PLATO)",
abstract = "Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA molecules to the proteins they encode, and recovered mRNA from affinity enrichment is subjected to analysis using massively parallel DNA sequencing. Compared with alternative in vitro methods, PLATO provides several advantages including library size and cost. A unique advantage of PLATO is that an alternative reverse transcription-quantitative PCR (RT-qPCR) protocol can be used to test binding of specific, individual proteins. To illustrate a typical experimental workflow, we demonstrate PLATO for the identification of the immune target of serum antibodies from patients with inclusion body myositis (IBM). Beginning with an ORFeome library in an RD vector, the protocol can produce samples for deep sequencing or RT-qPCR within 4 d.",
author = "Harry Larman and Liang, {Anthony C.} and Elledge, {Stephen J.} and Jian Zhu",
year = "2014",
month = "1",
doi = "10.1038/nprot.2013.167",
language = "English (US)",
volume = "9",
pages = "90--103",
journal = "Nature Protocols",
issn = "1754-2189",
publisher = "Nature Publishing Group",
number = "1",

}

TY - JOUR

T1 - Discovery of protein interactions using parallel analysis of translated ORFs (PLATO)

AU - Larman, Harry

AU - Liang, Anthony C.

AU - Elledge, Stephen J.

AU - Zhu, Jian

PY - 2014/1

Y1 - 2014/1

N2 - Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA molecules to the proteins they encode, and recovered mRNA from affinity enrichment is subjected to analysis using massively parallel DNA sequencing. Compared with alternative in vitro methods, PLATO provides several advantages including library size and cost. A unique advantage of PLATO is that an alternative reverse transcription-quantitative PCR (RT-qPCR) protocol can be used to test binding of specific, individual proteins. To illustrate a typical experimental workflow, we demonstrate PLATO for the identification of the immune target of serum antibodies from patients with inclusion body myositis (IBM). Beginning with an ORFeome library in an RD vector, the protocol can produce samples for deep sequencing or RT-qPCR within 4 d.

AB - Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA molecules to the proteins they encode, and recovered mRNA from affinity enrichment is subjected to analysis using massively parallel DNA sequencing. Compared with alternative in vitro methods, PLATO provides several advantages including library size and cost. A unique advantage of PLATO is that an alternative reverse transcription-quantitative PCR (RT-qPCR) protocol can be used to test binding of specific, individual proteins. To illustrate a typical experimental workflow, we demonstrate PLATO for the identification of the immune target of serum antibodies from patients with inclusion body myositis (IBM). Beginning with an ORFeome library in an RD vector, the protocol can produce samples for deep sequencing or RT-qPCR within 4 d.

UR - http://www.scopus.com/inward/record.url?scp=84891720952&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84891720952&partnerID=8YFLogxK

U2 - 10.1038/nprot.2013.167

DO - 10.1038/nprot.2013.167

M3 - Article

C2 - 24336473

AN - SCOPUS:84891720952

VL - 9

SP - 90

EP - 103

JO - Nature Protocols

JF - Nature Protocols

SN - 1754-2189

IS - 1

ER -