Patent ID: 6331400
Filing Date: 2001-12-18
Classification: G01N,Y10T

Abstract:
A method of identifying the position of a peptide amide within a sequence of a binding protein, said method comprising the steps of:(a) on-exchanging heavy hydrogen onto the binding protein for a period of time sufficient to label solvent-accessible peptide amides with said heavy hydrogen, to produce a first heavy hydrogen labeled binding protein and a second heavy hydrogen labeled binding protein;(b) off-exchanging solvent-accessible heavy hydrogen from said first heavy hydrogen labeled binding protein to produce a control and, under a same set of conditions, off-exchanging solvent-accessible heavy hydrogen from a complex between a binding partner and said second heavy hydrogen labeled binding protein, thereby producing, a functionally labeled binding protein that has at least one labeled peptide amide having a hydrogen exchange rate that is slowed in said complex compared to said exchange rate in said first heavy hydrogen labeled binding protein;(c) progressively degrading said control and said functionally labeled binding protein to obtain a series of sub-fragments for said control and a series of sub-fragments for said functionally labeled binding protein, wherein each sub-fragment is shorter than the corresponding preceding sub-fragment in each of said series by a single amino acid residue; and(d) determining which of said sub-fragments from said functionally labeled binding, protein are labeled with heavy hydrogen in comparison to said control, thereby identifying the position of each of said labeled peptide amides within the sequence of said binding proteinwherein steps (c) and (d) are carried out under conditions of slowed hydrogen exchange.