Mass spectrometric detection of affinity purified crosslinked peptides.
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.
Hurst
Gregory B
GB
Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6131, USA. hurstgb@ornl.gov
Lankford
Trish K
TK
Kennel
Stephen J
SJ
D000818:Animals; D001360:Avidin / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000737:chemistry*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D003432:Cross-Linking Reagents; D010455:Peptides; D001360:Avidin