Chemical Cross-linking and MALDI-TOF/TOF to Investigate Protein Interacting Domains

Abstract

INTRODUCTION: Mapping peptides modified by chemical cross-linker(s) provides clues about proteins' interacting domains. One complication is that such modification may result from intra- but not inter-molecular interactions. Western blot analyses showed an ability of pGSN to spontaneously form homo-dimers and homo-trimers, which we also detected in samples of human plasma. Therefore, main goal of this study was to use chemical cross-linking to map residues involved in inter- and/or intra-molecular interactions. METHODS: Two separate commercially available preparations were used in this study: recombinant expressed in E.coli (Cytoskeleton Inc.) and native purified from human plasma (Sigma, Inc.). Cross-linkers, Bis(sulfosuccinimidyl)glutarate-d⁰ (BS²G-d₀), Bis(sulfo-succinimidyl)suberate-d₀ (BS³G-d₀), Bis-N-succinimidyl(PEG)₅ (BS(PEG)⁵) and 3,3′-Dithiobis (sulfosuccinimidylpropionate) (DTSSP) were from Thermo Scientific (San Jose, CA, USA). For mass spectrometry analyses we used ABSciex 4800 MALDI-TOF/TOF. RESULTS: We used cross-linkers with arm length ranging from 7.7Å to 21.7Å. We present that MALDI based mass spectrometry generates high quality data to show lysine residues modified by cross-linkers and combined with existing data based on crystallography (Protein Data Bank, PDB) can be used to discriminate between inter- and intra-molecular linking. Interestingly, a 12.0Å linker did not support complete intra-molecular cross-linking of monomer like 11.4Å cross-linker resulting in the presence of two bands in 1-DE analysis. CONCLUSIONS: When interpreting results of in vitro cross-linking interacting proteins, a possibility of self cross-linking of any of interacting proteins should be considered. Therefore, critical residues for forming hetero-complexes can be blocked and new and not biologically relevant interacting domains can be created. Flexibility of some regions within the protein polypeptide chain may lead to linking of distant residues where linker length is half the distance thus such assumption cannot be made. Non-cleavable cross-linkers provided confirmation of intra-molecular cross-linking, while cleavable cross-linker did not. The manual interpretation of MS/MS spectra allowed us to gather more specific information.