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

作者：Chakrabarty Jayanta K; Naik Aishwarya G; Fessler Michael B; Munske Gerhard R; Chowdhury Saiful M^*

来源：Analytical Chemistry, 2016, 88(20): 10215-10222.

DOI：10.1021/acs.analchem.6b02886

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.

• 出版日期2016-10-18