Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states.

Dan Tan,Pan Zhang,Qiang Li,Chao Liu,Ning Gao,Yue-He Ding,Xiaoguang Lei,Bing Yang,Xiangke Li,Xiaohui Liu,Boya Feng,Shoucai Ma,Junjie Liu,Keqiong Ye,Ли Тао ,Hongwei Wang ,Meijun Zhang ,Meng‐Qiu Dong ,Shan He ,Shidong Fan ,Chunyang Ma

doi:10.7554/elife.12509

Abstract

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.

Highlights

Proteins execute diverse functions by interacting with multiple protein partners in different complexes
We further extended the application of quantitative CXMS to a highly complex system consisting of log-phase and stationary-phase E. coli cells and identified a growth phase specific protein interaction
We aimed to develop a cross-linker similar to the widely used BS3 but that had two major advantages: first, a biotin tag for affinity purification of cross-linked peptides, and second, a cleavage site to release cross-linked peptides after enrichment on streptavidin beads without carrying the biotin group; biotin can interfere with subsequent LC-MS/MS analysis

Summary

Introduction

Proteins execute diverse functions by interacting with multiple protein partners in different complexes. The study of protein complex structures and protein-protein interactions is critical for understanding their functions. Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) has emerged as a powerful tool for the analysis of such structures and interactions (Sinz, 2006; Leitner et al, 2010; Petrotchenko and Borchers, 2010; Singh et al, 2010; Rappsilber, 2011; Bruce, 2012). Recent progress in the development of analytical instruments, cross-linking reagents, and software has catapulted CXMS from obscurity to prominence, as witnessed by an explosion of successful applications

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