Retracted: Isocratic Method for Affinity Enrichment of Covalently‐linked Peptides in Cyanogen Bromide Cleavage of Proteins

The low resolution three-dimensional structure of a protein can be inferred from existing disulfide bridges or experimentally introduced chemical crosslinks. The general procedure involves enzymatic digestion of a protein followed by mass spectrometry-based identification of covalently-linked peptides, native disulfide-linked peptides and chemically cross-linked peptides. To facilitate unambiguous identification of these peptides, an isocratic purification method was developed for selective enrichment of covalently-linked cyanogen bromide (CNBr) fragments. This method capitalizes on the ability of homoserine lactone moieties at the C-termini of CNBr cleavage products for selective conjugation of primary-amine containing affinity tag. The availability of two C-termini within covalently-linked peptides allows for the conjugation of two affinity tags, whereas the other peptides have only one affinity tag at the C-terminus, which enables selective enrichment of covalently-linked peptides by utilization of affinity tag with moderate dissociation constant. Here we demonstrate successful implementation of this method with tetrahistidine as the affinity tag for enrichment of covalently-linked CNBr fragments of test peptides and proteins.