Patent ID: 7166436
Filing Date: 2007-01-23
Classification: C07K,G01N

Abstract:
1. A method for simultaneously identifying and determining the levels of expression of cysteine-containing proteins in normal and perturbed cells, comprising: (a) preparing a first protein sample from a normal cell; (b) reacting the first protein sample with a first reagent consisting of an amino acid sequence selected from the group consisting of Acyl-NH-CASENLYFQG-Lys-ε-iodoacetamide (SEQ ID NO:41), Acyl-NH-CASENLYFQG-Orn-δ-iodoacetamide (SEQ ID NO:42), Acyl-NH-CASENLYFQGP-Lys-ε-iodoacetamide (SEQ ID NO:43), and Acyl-NH-CASENLYFQGP-Orn-δ-iodoacetamide (SEQ ID NO:44); (c) preparing a second protein sample from a perturbed cell; (d) reacting the second protein sample of step (c) with a second reagent consisting of an amino acid sequence selected from the group consisting of Acyl-NH-CASENLYFQG-Lys-ε-iodoacetamide (SEQ ID NO:41), Acyl-NH-CASENLYFQG-Orn-δ-iodoacetamide (SEQ ID NO:42), Acyl-NH-CASENLYFQGP-Lys-ε-iodoacetamide (SEQ ID NO:43), and Acyl-NH-CASENLYFQGP-Orn-δ-iodoacetamide (SEQ ID NO:44), (e) combining the reacted the first and the second protein samples from steps (b) and (d); (f) subjecting the combined protein samples from step (e) to proteolysis at a site on the protein samples, the site being other than a Protease Cleavage Site, wherein the Protease Cleavage Site is an amino acid sequence of SEQ ID NO:1 that is a cleavage site for TEV protease; (g) subjecting the proteolyzed combined protein samples from step (f) to an affinity chromatography system comprising an amino acid sequence attached to a solid, thereby forming bound proteins and non-bound proteins, (h) eluting the non-bound proteins from the affinity chromatography system; (i) subjecting the affinity chromatography system from step (h) to a TEV protease, thereby forming a cleaved protein mixture; (j) eluting the cleaved protein mixture from the affinity chromatography system of step i); (k) isolating the eluted protein mixture from step (j); (l) subjecting the eluted protein mixture from step (k) to chromatographic separation, followed by mass analysis; (m) determining from the results of step (l) a ratio of amounts of the cysteine-containing proteins from the normal cells reacted with the first reagent and the cysteine-containing proteins from the perturbed cells reacted with the second reagent, where the molecular weights thereof are separated by an integer multiple of 14 atomic mass units; and (n) comparing the results obtained for each cysteine-containing protein to protein databases containing chromatographic and molecular weight correlations to identify the cysteine-containing proteins.