Proteomics Lecture 6 Proteases 3

Protein can be cleaved by chemicals. The most widely used of these is Cyanogen Bromide (CNBr). Although the reaction proceeds with a high degree of specificity, the relative infrequency of methionine residues in the most proteins means that CNBr cleavage yields relatively few, large fragments.

Cyanogen bromide is a pseudohalogen compound with the formula CNBr. It is a colorless solid that is widely used to modify biopolymers, fragment proteins and peptides, and synthesize other compounds. Cyanogen bromide hydrolyzes peptide bonds at the C-terminus of methionine residues. This reaction is used to reduce the size of polypeptide segments for identification and sequencing.

Mechanism In CNBr, the electron density is shifted away from the carbon atom, making it unusually electrophilic, and towards the more electronegative bromine and nitrogen. This leaves the carbon particularly vulnerable to attack by a nucleophile, and the cleavage reaction begins with a nucleophilic acyl substitution reaction in which bromine is ultimately replaced by the sulfur in methionine. This attack is followed by the formation of a five-membered ring as opposed to a six-membered ring, which would entail the formation of a double bond in the ring between nitrogen and carbon. This double bond would result in a rigid ring conformation, thereby destabilizing the molecule. Thus, the five-membered ring is formed so that the double bond is outside the ring. Although the nucleophilic sulfur in methionine is responsible for attacking CNBr, the sulfur in cysteine does not behave similarly. The strongest electrophile would then be the cyanide nitrogen, which, if attacked by water, would yield cyanic acid and the original cysteine. (http://en.wikipedia.org/wiki/Cyanogen_bromide)

In-Gel Digestion Protein separated by 1 D or 2 D-SDS PAGE referred to as in-gel digestion. The band or spot of interest is cut from the gel, distained, and then treated with protease. The enzyme penetrates the gel matrix and digests the protein to peptides which then eluted from the gel by washing.