A Universal Peptide Matrix Interactomics Approach to Disclose Motif-Dependent Protein Binding

Autor: Ramberger, Evelyn, Suarez-Artiles, Lorena, Perez-Hernandez, Daniel, Haji, Mohamed, Popp, Oliver, Reimer, Ulf, Leutz, Achim, Dittmar, Gunnar, Mertins, Philipp
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Proteomics
FDR
false discovery rate
point mutations
Amino Acid Motifs
PTMs
post-translational modifications
PPIs
protein–protein interactions
IDRs
intrinsically disordered regions
SLiMs
short linear motifs
post-translational modifications
Humans
Point Mutation
AGC
automated gain control
Protein Interaction Domains and Motifs
TCEP
Tris(2-carboxyethyl)phosphine hydrochloride
ABC
ammonium bicarbonate
MBR
match between run
Technological Innovation and Resources
LFQ
label-free quantification
short linear motifs
CAA
chloroacetamide
MoRFs
molecular recognition features
EGFR
epidermal growth factor receptor
peptide interaction screen
CRs
conserved regions
PRISMA
protein interaction screen on a peptide matrix
IT
injection time
Peptides
Protein Processing
Post-Translational
SH2
Src homology 2
HeLa Cells
Protein Binding
Zdroj: Molecular & Cellular Proteomics : MCP
ISSN: 1535-9484
1535-9476
Popis: Protein–protein interactions mediated by intrinsically disordered regions are often based on short linear motifs (SLiMs). SLiMs are implicated in signal transduction and gene regulation yet remain technically laborious and notoriously challenging to study. Here, we present an optimized method for a protein interaction screen on a peptide matrix (PRISMA) in combination with quantitative MS. The protocol was benchmarked with previously described SLiM-based protein–protein interactions using peptides derived from EGFR, SOS1, GLUT1, and CEBPB and extended to map binding partners of kinase activation loops. The detailed protocol provides practical considerations for setting up a PRISMA screen and subsequently implementing PRISMA on a liquid-handling robotic platform as a cost-effective high-throughput method. Optimized PRISMA can be universally applied to systematically study SLiM-based interactions and associated post-translational modifications or mutations to advance our understanding of the largely uncharacterized interactomes of intrinsically disordered protein regions.
Graphical Abstract
Highlights • Optimized protocol for analysis of peptide–protein interactions with peptide arrays. • Detection of interactions affected by mutations or post-translational modifications. • Mapping of interaction sites with overlapping peptide sequences. • Implementation on a liquid-handling robotic platform.
In Brief A universal and optimized peptide matrix pull-down approach enables cost-effective and reproducible high-throughput interactome analysis of peptides with reduced analysis time. Our data provide proof of principle for the wide applicability of our streamlined protocol to map and detect motif-based protein interactions influenced by mutations and post-translational modifications.
Databáze: OpenAIRE
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