Differential Tandem Mass Spectrometry-Based Cross-Linker: A New Approach for High Confidence in Identifying Protein Cross-Linking

被引:23
|
作者
Chakrabarty, Jayanta K. [1 ]
Naik, Aishwarya G.
Fessler, Michael B. [2 ]
Munske, Gerhard R. [3 ]
Chowdhury, Saiful M. [1 ]
机构
[1] Univ Texas Arlington, Dept Chem & Biochem, 700 Planetarium Pl, Arlington, TX 76019 USA
[2] NIEHS, Immun Inflammat & Dis Lab, NIH, POB 12233, Res Triangle Pk, NC 27709 USA
[3] Washington State Univ, Lab Bioanal, Pullman, WA 98195 USA
关键词
MAMMALIAN MITOCHONDRIAL RIBOSOME; ELECTRON-TRANSFER DISSOCIATION; IDENTIFICATION; PEPTIDES; METHODOLOGY; REAGENTS; STRATEGY; BONDS;
D O I
10.1021/acs.analchem.6b02886
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Chemical cross-linking and mass spectrometry are now widely used to analyze large-scale protein-protein interactions. The major challenge in cross-linking approaches is the complexity of the mass spectrometric data. New approaches are required that can identify cross-linked peptides with high-confidence and establish a user-friendly analysis protocol for the biomedical scientific community. Here, we introduce a novel cross-linker that can be selectively cleaved in the gas phase using two differential tandem mass-spectrometric fragmentation methods, such as collision-induced or electron transfer dissociation (CID and ETD). This technique produces two signature mass spectra of the same cross-linked peptide, thereby producing high confidence in identifying the sites of interaction. Further-tandem mass spectrometry can also give additional confidence on the peptide sequences. We demonstrate a proof-of-concept for This method using standard peptides and proteins. Peptides and proteins were cross-linked and their fragmentation characteristics were analyzed using CID and ETD tandem mass spectrometry. Two sequential cleavages unambiguously identified cross-linked peptides. In addition, the labeling efficiency of the new cross-linker was evaluated in macrophage immune cells after stimulation with the microbial ligand lipopolysaccharide and subsequent pulldown experiments with biotin-avidin affinity chromatography. We believe this strategy will help advance insights into the structural biology and systems biology of cell signaling.
引用
收藏
页码:10215 / 10222
页数:8
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