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Molecular & Cellular Proteomics 8:273-286, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

BiPS, a Photocleavable, Isotopically Coded, Fluorescent Cross-linker for Structural Proteomics ^*

Evgeniy V. Petrotchenko, Kunhong Xiao^,¶, Jennifer Cable, Yiwen Chen, Nikolay V. Dokholyan and Christoph H. Borchers^,||

From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599 and Department of Medicine, Duke University, Durham, North Carolina 27710

Cross-linking combined with mass spectrometry is an emerging approach for studying protein structure and protein-protein interactions. However, unambiguous mass spectrometric identification of cross-linked peptides derived from proteolytically digested cross-linked proteins is still challenging. Here we describe the use of a novel cross-linker, bimane bisthiopropionic acid N-succinimidyl ester (BiPS), that overcomes many of the challenges associated with other cross-linking reagents. BiPS is distinguished from other cross-linkers by a unique combination of properties: it is photocleavable, fluorescent, homobifunctional, amine-reactive, and isotopically coded. As demonstrated with a model protein complex, RNase S, the fluorescent moiety of BiPS allows for sensitive and specific monitoring of the different cross-linking steps, including detection and isolation of cross-linked proteins by gel electrophoresis, determination of in-gel digestion completion, and fluorescence-based separation of cross-linked peptides by HPLC. The isotopic coding of BiPS results in characteristic ion signal "doublets" in mass spectra, thereby permitting ready detection of cross-linker-containing peptides. Under MALDI-MS conditions, partial photocleavage of the cross-linker occurs, releasing the cross-linked peptides. This allows differentiation between dead-end, intra-, and interpeptide cross-links based on losses of specific mass fragments. It also allows the use of the isotope doublets as mass spectrometric "signatures." A software program was developed that permits automatic cross-link identification and assignment of the cross-link type. Furthermore photocleavage of BiPS assists in cross-link identification by allowing separate tandem mass spectrometry sequencing of each peptide comprising the original cross-link. By combining the use of BiPS with MS, we have provided the first direct evidence for the docking site of a phosphorylated G-protein-coupled receptor C terminus on the multifunctional adaptor protein β-arrestin, clearly demonstrating the broad potential and application of this novel cross-linker in structural and cellular biology.

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