Profiling phosphopeptide-binding domain recognition specificity using peptide microarrays

Michele Tinti (Lead / Corresponding author), Simona Panni (Lead / Corresponding author), Gianni Cesareni

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

1 Citation (Scopus)

Abstract

Cellular organization and response to internal and external stimuli are mediated by an intricate web of protein interactions. Some of these interactions are regulated by covalent posttranslational modifications such as phosphorylation and acetylation. These modifications can change the chemical nature of the interaction interfaces and modulate the binding affinity of the interacting partners. In signal transduction, the most frequent modification is reversible phosphorylation of tyrosine, serine or threonine residues. Protein phosphorylation may modulate the activity of enzymes by modifying their conformation, or regulate the formation of complexes by creating docking sites to recruit downstream effectors. Families of modular domains, such as SH2, PTB, and 14-3-3, act as "readers" of the modification event. Specificity between closely related domains of the same family is mediated by the chemical properties of the domain binding surface that, aside from offering a hydrophilic pocket for the phosphorylated residue, shows preference for specific sequences. Although the protein structure and the cell context are also important to ensure specificity, the amino acid sequence flanking the phosphorylation site defines the accuracy of the recognition process, and it is therefore essential to define the binding specificity of phosphopeptide binding domains in order to understand and to infer the interaction web mediated by phosphopeptides. Methods commonly used to discover new interactions (such as yeast two hybrid and phage display) are not suited to study interactions with phosphorylated proteins. On the other hand, peptide arrays are a powerful approach to precisely identify the binding preference of phosphopeptide recognition domains. Here we describe a detailed protocol to assemble arrays of hundreds to thousands phospho-peptides and to screen them with any modular domain of interest.

Original languageEnglish
Title of host publicationSmall molecule microarrays
Subtitle of host publicationmethods and protocols
EditorsMahesh Uttamchandani , Shao Q. Yao
Place of PublicationNew York
PublisherSpringer
Pages177-193
Number of pages17
ISBN (Electronic)9781493965847
ISBN (Print)9781493965823
DOIs
Publication statusPublished - 2017

Publication series

NameMethods in molecular biology
PublisherSpringer
Volume1518
ISSN (Print)1064-3745

Fingerprint

Phosphopeptides
Phosphorylation
Peptides
Proteins
Threonine
Post Translational Protein Processing
Acetylation
Bacteriophages
Serine
Tyrosine
Amino Acid Sequence
Signal Transduction
Yeasts
Enzymes

Keywords

  • Phosphopeptide
  • Spot synthesis
  • 14-3-3
  • SH2
  • Interaction networks
  • Binding domains

Cite this

Tinti, M., Panni, S., & Cesareni, G. (2017). Profiling phosphopeptide-binding domain recognition specificity using peptide microarrays. In M. Uttamchandani , & S. Q. Yao (Eds.), Small molecule microarrays: methods and protocols (pp. 177-193). (Methods in molecular biology; Vol. 1518). New York: Springer . https://doi.org/10.1007/978-1-4939-6584-7_12
Tinti, Michele ; Panni, Simona ; Cesareni, Gianni. / Profiling phosphopeptide-binding domain recognition specificity using peptide microarrays. Small molecule microarrays: methods and protocols. editor / Mahesh Uttamchandani ; Shao Q. Yao. New York : Springer , 2017. pp. 177-193 (Methods in molecular biology).
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Tinti, M, Panni, S & Cesareni, G 2017, Profiling phosphopeptide-binding domain recognition specificity using peptide microarrays. in M Uttamchandani & SQ Yao (eds), Small molecule microarrays: methods and protocols. Methods in molecular biology, vol. 1518, Springer , New York, pp. 177-193. https://doi.org/10.1007/978-1-4939-6584-7_12

Profiling phosphopeptide-binding domain recognition specificity using peptide microarrays. / Tinti, Michele (Lead / Corresponding author); Panni, Simona (Lead / Corresponding author); Cesareni, Gianni.

Small molecule microarrays: methods and protocols. ed. / Mahesh Uttamchandani ; Shao Q. Yao. New York : Springer , 2017. p. 177-193 (Methods in molecular biology; Vol. 1518).

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

TY - CHAP

T1 - Profiling phosphopeptide-binding domain recognition specificity using peptide microarrays

AU - Tinti, Michele

AU - Panni, Simona

AU - Cesareni, Gianni

PY - 2017

Y1 - 2017

N2 - Cellular organization and response to internal and external stimuli are mediated by an intricate web of protein interactions. Some of these interactions are regulated by covalent posttranslational modifications such as phosphorylation and acetylation. These modifications can change the chemical nature of the interaction interfaces and modulate the binding affinity of the interacting partners. In signal transduction, the most frequent modification is reversible phosphorylation of tyrosine, serine or threonine residues. Protein phosphorylation may modulate the activity of enzymes by modifying their conformation, or regulate the formation of complexes by creating docking sites to recruit downstream effectors. Families of modular domains, such as SH2, PTB, and 14-3-3, act as "readers" of the modification event. Specificity between closely related domains of the same family is mediated by the chemical properties of the domain binding surface that, aside from offering a hydrophilic pocket for the phosphorylated residue, shows preference for specific sequences. Although the protein structure and the cell context are also important to ensure specificity, the amino acid sequence flanking the phosphorylation site defines the accuracy of the recognition process, and it is therefore essential to define the binding specificity of phosphopeptide binding domains in order to understand and to infer the interaction web mediated by phosphopeptides. Methods commonly used to discover new interactions (such as yeast two hybrid and phage display) are not suited to study interactions with phosphorylated proteins. On the other hand, peptide arrays are a powerful approach to precisely identify the binding preference of phosphopeptide recognition domains. Here we describe a detailed protocol to assemble arrays of hundreds to thousands phospho-peptides and to screen them with any modular domain of interest.

AB - Cellular organization and response to internal and external stimuli are mediated by an intricate web of protein interactions. Some of these interactions are regulated by covalent posttranslational modifications such as phosphorylation and acetylation. These modifications can change the chemical nature of the interaction interfaces and modulate the binding affinity of the interacting partners. In signal transduction, the most frequent modification is reversible phosphorylation of tyrosine, serine or threonine residues. Protein phosphorylation may modulate the activity of enzymes by modifying their conformation, or regulate the formation of complexes by creating docking sites to recruit downstream effectors. Families of modular domains, such as SH2, PTB, and 14-3-3, act as "readers" of the modification event. Specificity between closely related domains of the same family is mediated by the chemical properties of the domain binding surface that, aside from offering a hydrophilic pocket for the phosphorylated residue, shows preference for specific sequences. Although the protein structure and the cell context are also important to ensure specificity, the amino acid sequence flanking the phosphorylation site defines the accuracy of the recognition process, and it is therefore essential to define the binding specificity of phosphopeptide binding domains in order to understand and to infer the interaction web mediated by phosphopeptides. Methods commonly used to discover new interactions (such as yeast two hybrid and phage display) are not suited to study interactions with phosphorylated proteins. On the other hand, peptide arrays are a powerful approach to precisely identify the binding preference of phosphopeptide recognition domains. Here we describe a detailed protocol to assemble arrays of hundreds to thousands phospho-peptides and to screen them with any modular domain of interest.

KW - Phosphopeptide

KW - Spot synthesis

KW - 14-3-3

KW - SH2

KW - Interaction networks

KW - Binding domains

U2 - 10.1007/978-1-4939-6584-7_12

DO - 10.1007/978-1-4939-6584-7_12

M3 - Chapter (peer-reviewed)

SN - 9781493965823

T3 - Methods in molecular biology

SP - 177

EP - 193

BT - Small molecule microarrays

A2 - Uttamchandani , Mahesh

A2 - Yao, Shao Q.

PB - Springer

CY - New York

ER -

Tinti M, Panni S, Cesareni G. Profiling phosphopeptide-binding domain recognition specificity using peptide microarrays. In Uttamchandani M, Yao SQ, editors, Small molecule microarrays: methods and protocols. New York: Springer . 2017. p. 177-193. (Methods in molecular biology). https://doi.org/10.1007/978-1-4939-6584-7_12