Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β 1

Abstract

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β 1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β 1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β 1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β 1, α 4 and α 6 or β 1, α 6, α 2, and α 5, respectively.

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