Abstract
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 beta-arrestin, clearly demonstrating the broad potential and application of this novel cross-linker in structural and cellular biology.
Highlights
Cross-linking combined with mass spectrometry is an emerging approach for studying protein structure and protein-protein interactions
Design and Analytical Characteristics of the Fluorescent, Photocleavable, and Isotopically Coded Cross-linker bisthiopropionic acid Nsuccinimidyl ester (BiPS)—In previous studies, we found that the modification of cysteine residues by MALDI-cleavable Cross-linker for Structural Proteomics bimane (MBB), a commercially available fluorescent label for thiol groups, was partially reversible during MALDI-MS [10]
BiPS is distinguished from other reagents by a unique combination of chemical groups that render it homobifunctional, amine-reactive, isotopically coded, fluorescent, and photocleavable under MALDI-MS conditions
Summary
Design and Analytical Characteristics of the Fluorescent, Photocleavable, and Isotopically Coded Cross-linker BiPS—In previous studies, we found that the modification of cysteine residues by MBB, a commercially available fluorescent label for thiol groups, was partially reversible during MALDI-MS [10]. When combined with GPS ExplorerTM or Mascot analysis of MS/MS data for individual peptides obtained after cross-linker cleavage, ICC-CLASS can be used to identify unknown dead-end, inter-, and intrapeptide cross-links This program is designed as an integrated software/instrument platform allowing a completely automated and targeted analysis of isotopically coded cross-links from data acquisition by MALDI-MS and -MS/MS to the assignment and identification of the cross-links. Detailed examination of the x-ray crystallographic structure of RNase S S-protein and S-peptide binding sites revealed that the structural information obtained from the cross-linked peptides agrees well with our protein-protein interaction site data because the distances between cross-linked sites are within the maximum span of the cross-linker [26] These experiments demonstrate the feasibility of using BiPS to structurally characterize protein-protein interaction sites as well as the capability of ICC-CLASS to facilitate detection, assignment, and identification of cross-links. These data are in agreement with the current receptor-arrestin
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