| US 7,544,518 B2 | ||
| Rapid quantitative analysis of proteins or protein function in complex mixtures | ||
| Rudolf Hans Aebersold, Mercer Island, Wash. (US); Michael H. Gelb, Seattle, Wash. (US); Steven P. Gygi, Seattle, Wash. (US); C. Ronald Scott, Seattle, Wash. (US); Frantisek Turecek, Seattle, Wash. (US); Scott A. Gerber, Seattle, Wash. (US); and Beate Rist, Seattle, Wash. (US) | ||
| Assigned to University of Washington, Seattle, Wash. (US) | ||
| Filed on Nov. 23, 2004, as Appl. No. 10/994,815. | ||
| Application 10/994815 is a continuation of application No. 09/839884, filed on Apr. 20, 2001, granted, now 6,852,544. | ||
| Application 09/839884 is a continuation of application No. 09/383062, filed on Aug. 25, 1999, granted, now 6,670,194. | ||
| Claims priority of provisional application 60/099113, filed on Sep. 03, 1998. | ||
| Claims priority of provisional application 60/097788, filed on Aug. 25, 1998. | ||
| Prior Publication US 2005/0233399 A1, Oct. 20, 2005 | ||
| This patent is subject to a terminal disclaimer. | ||
| Int. Cl. G01N 33/68 (2006.01) | ||
| U.S. Cl. 436—173 [436/86; 436/89; 436/120; 436/161; 436/165; 436/166; 436/167; 436/168; 436/171; 436/174; 436/175; 436/177; 530/350; 530/812; 530/391.5] | 73 Claims |
| 1. A method for identifying one or more enzymes in one or more samples containing mixtures of enzymes which comprises the
steps:
(a) providing affinity tagged protein reactive reagents wherein the reagents have the formula:
A-L-PRG
where A is an affinity label that selectively binds to a capture reagent, L is a linker group in which one or more atoms
is differentially labeled with one or more stable isotopes and PRG is a protein reactive group that is a substrate for an
enzyme, wherein an affinity tagged protein reactive reagent is provided for each enzyme that is to be detected and identified
in each sample by differentially labeling the linker group of the affinity tagged protein reactive reagent with one or more
stable isotopes;
(b) reacting each sample with the protein reactive reagents to provide affinity tagged differentially labeled enzyme products
in each sample;
(c) capturing any affinity tagged differentially labeled enzyme products in the samples using the capture reagent that selectively
binds A;
(d) releasing captured affinity tagged differentially labeled enzyme products from the capture reagent by disrupting the interaction
between the affinity tagged differentially labeled enzyme products and the capture reagent; and
(e) detecting and identifying the released affinity tagged differentially labeled enzyme products by mass spectrometry to
thereby identify one or more enzymes in the one or more samples.
|