Mass spectrometric detection of affinity purified crosslinked peptides

Gregory B. Hurst, Trish K. Lankford, Stephen Kennel

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest of the products shows considerable promise as a tool for interrogating structure and geometry of proteins and protein complexes. An impediment to the use of this tool has been the difficulty of distinguishing crosslinked peptide pairs from non-crosslinked peptides, and from the products of side reactions. We describe the use of a commercially available biotinylated crosslinking reagent, sulfo-SBED, that allows affinity-based enrichment of crosslinked species. An intramolecular crosslink is prepared using the peptide neurotensin as a model system. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra show the predicted crosslinking product, as well as several side products. Finally, we describe the optimized enrichment of biotinylated species, and reduction of non-specific binding, for a batch-mode affinity separation based on immobilized monomeric avidin.

Original languageEnglish (US)
Pages (from-to)832-839
Number of pages8
JournalJournal of the American Society for Mass Spectrometry
Volume15
Issue number6
DOIs
StatePublished - Jun 1 2004
Externally publishedYes

Fingerprint

Crosslinking
Peptides
Cross-Linking Reagents
Neurotensin
Proteins
Avidin
Ionization
Desorption
Lasers
Geometry

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Spectroscopy

Cite this

Mass spectrometric detection of affinity purified crosslinked peptides. / Hurst, Gregory B.; Lankford, Trish K.; Kennel, Stephen.

In: Journal of the American Society for Mass Spectrometry, Vol. 15, No. 6, 01.06.2004, p. 832-839.

Research output: Contribution to journalArticle

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