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Collision-induced dissociative chemical cross-linking reagents and methodology: Applications to protein structural characterization using tandem mass spectrometry analysis.

Publication ,  Journal Article
Soderblom, EJ; Goshe, MB
Published in: Anal Chem
December 1, 2006

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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Published In

Anal Chem

DOI

ISSN

0003-2700

Publication Date

December 1, 2006

Volume

78

Issue

23

Start / End Page

8059 / 8068

Location

United States

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
 
Journal cover image

Published In

Anal Chem

DOI

ISSN

0003-2700

Publication Date

December 1, 2006

Volume

78

Issue

23

Start / End Page

8059 / 8068

Location

United States

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