Collision-induced dissociative chemical cross-linking reagents and methodology: Applications to protein structural characterization using tandem mass spectrometry analysis.
Chemical cross-linking combined with mass spectrometry is a viable approach to study the low-resolution structure of protein and protein complexes. However, unambiguous identification of the residues involved in a cross-link remains analytically challenging. To enable a more effective analysis across various MS platforms, we have developed a novel set of collision-induced dissociative cross-linking reagents and methodology for chemical cross-linking experiments using tandem mass spectrometry (CID-CXL-MS/MS). These reagents incorporate a single gas-phase cleavable bond within their linker region that can be selectively fragmented within the in-source region of the mass spectrometer, enabling independent MS/MS analysis for each peptide. Initial design concepts were characterized using a synthesized cross-linked peptide complex. Following verification and subsequent optimization of cross-linked peptide complex dissociation, our reagents were applied to homodimeric glutathione S-transferase and monomeric bovine serum albumin. Cross-linked residues identified by our CID-CXL-MS/MS method were in agreement with published crystal structures and previous cross-linking studies using conventional approaches. Common LC/MS/MS acquisition approaches such as data-dependent acquisition experiments using ion trap mass spectrometers and product ion spectral analysis using SEQUEST were shown to be compatible with our CID-CXL-MS/MS reagents, obviating the requirement for high resolution and high mass accuracy measurements to identify both intra- and interpeptide cross-links.
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Related Subject Headings
- Tandem Mass Spectrometry
- Serum Albumin, Bovine
- Schistosoma japonicum
- Recombinant Fusion Proteins
- Protein Structure, Tertiary
- Proline
- Molecular Sequence Data
- Models, Molecular
- Glutathione Transferase
- Cross-Linking Reagents
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Tandem Mass Spectrometry
- Serum Albumin, Bovine
- Schistosoma japonicum
- Recombinant Fusion Proteins
- Protein Structure, Tertiary
- Proline
- Molecular Sequence Data
- Models, Molecular
- Glutathione Transferase
- Cross-Linking Reagents