Journal Article

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

Zhang, Pan, Gao, Ning, Liu, Chao, Yang, Bing, Lei, Xiaoguang, Wang, Hong-Wei, Ye, Keqiong, Fan, Sheng-Bo, Feng, Boya, Liu, Xiaohui, He, Si-Min, Li, Qiang, Tao, Li, Tan, Dan, Zhang, Mei-Jun, Li, Xiangke, Dong, Meng-Qiu, Ma, Chengying, Ding, Yue-He, Ma, Shoucai, Liu, Junjie

2016

Overview

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. Proteins fold into structures that are determined by the order of the amino acids that they are built from. These structures enable the protein to carry out its role, which often involves interacting with other proteins. Chemical cross-linking coupled with mass spectrometry (CXMS) is a powerful method used to study protein structure and how proteins interact, with a benefit of stabilizing and capturing brief interactions. CXMS uses a chemical compound called a linker that has two arms, each of which can bind specific amino acids in a protein or in multiple proteins. Only when the regions are close to each other can they be “cross-linked” in this way. After cross-linking, the proteins are cut into small pieces known as peptides. The cross-linked peptides are then separated from the non cross-linked ones and characterized. Although CXMS is a popular method, there are aspects about it that limit its use. It does not work well on complex samples that contain lots of different proteins, as it is difficult to separate the cross-linked peptides from the overwhelming amounts of non cross-linked peptides. Also, although it can be used to detect changes in the shape of a protein, which are often crucial to the protein's role, the method has not been smoothed out. Tan, Li et al. have now developed a new cross-linker called Leiker that addresses these limitations. Leiker cross-links the amino acid lysine to another lysine, and contains a molecular tag that allows cross-linked peptides to be efficiently purified away from non cross-linked peptides. As part of a streamlined workflow to detect changes in the shape of a protein, Leiker also contains a region that can be labeled. Analysing a bacterial ribosome, which contains more than 50 proteins, showed that Leiker-based CXMS could detect many more protein interactions than previous studies had. These included interactions that changed too rapidly to be studied by other structural methods. Tan, Li et al. then applied Leiker-based CXMS to the entire contents of bacterial cells at different stages of growth, and identified a protein interaction that is only found in growing cells. In future, Leiker will be useful for analyzing the structure of large protein complexes, probing changes in protein structure, and mapping the interactions between proteins in complex mixtures.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: 70S ribosome / Animals / Biophysics and Structural Biology / Biotin / Caenorhabditis elegans - chemistry / Caenorhabditis elegans - physiology / Caenorhabditis elegans Proteins - analysis / Caenorhabditis elegans Proteins - chemistry / cross-linking / Cross-Linking Reagents - metabolism / Crystallography / E coli / Electron microscopy / Escherichia coli - chemistry / Escherichia coli - physiology / Escherichia coli Proteins - analysis / Escherichia coli Proteins - chemistry / exosome / Identification / Isotopes / Life sciences / Lysates / Lysine / mass spectrometry / Mass spectroscopy / Nucleic acids / Peptide mapping / Peptides / Protein Conformation / Protein interaction / Protein Interaction Mapping - methods / Protein Interaction Maps / Protein purification / protein structure / protein-protein interactions / Proteins / Quantitation / Ratios / Ribosomes - chemistry / Software / Tools and Resources