Open AccessMass spectrometric detection of affinity purified crosslinked presented
Author(s) -
Gregory B. Hurst,
Trish K. Lankford,
Stephen J. Kennel
Publication year - 2004
Publication title -
journal of the american society for mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.961
H-Index - 127
eISSN - 1879-1123
pISSN - 1044-0305
DOI - 10.1016/j.jasms.2004.02.008
Subject(s) - chemistry , biotinylation , avidin , matrix assisted laser desorption/ionization , mass spectrometry , chromatography , peptide , combinatorial chemistry , reagent , desorption , monomer , polymer , organic chemistry , adsorption , biochemistry
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.