Diagnostic methods for ubiquinated protein profiling
View Patent ↗Methods for establishing a protein expression profile of a biological sample, by contacting a biological sample with a ubiquitin-binding protein such that ubiquitinated and specific non-ubiquitinated protein (the IPS) bind to the ubiquitin-binding protein; isolating the IPS proteins; and analyzing the isolated IPS proteins, wherein an expression profile is generated.
1. A method for establishing an expression profile of purified ubiquitinated proteins which are targeted for degradation by a ubiquitin pathway for a disease or pathological biological sample comprising ubiquitinated proteins, wherein the biological sample is a tissue, a cell, a peripheral blood sample, a body fluid sample, a tissue biopsy sample, protein extract isolated from the tissue or cell, or a stool sample, comprising
(a) contacting the biological sample comprising ubiquitinated proteins which are targeted for degradation by the ubiquitin pathway with a ubiquitin-specific-binding protein attached to an affinity matrix, wherein the ubiquitin-specific-binding protein consists of ubiquitin-associated domain 1 (UBAI) of Rad23, such that ubiquitinated proteins which are targeted for degradation by the ubiquitin pathway bind to the ubiquitin-specific-binding protein;
(b) separating ubiquitinated proteins that bound the ubiquitin-specific-binding protein in step (a) and which are targeted for degradation by the ubiquitin pathway from affinity matrix supernatant comprising proteins of the biological sample that did not bind to the ubiquitin-specific-binding protein in step (a);
(c) contacting the affinity matrix supernatant from step (b) with a ubiquitin-specific-bindingprotein attached to a new affinity matrix, wherein the ubiquitin-specific-binding protein also consists of ubiquitin-associated domain 1 (UBAI) of Rad23, such that ubiquitinated proteins which are targeted for degradation by the ubiquitin pathway bind to the ubiquitin-specific-bindingprotein;
(d) isolating ubiquitinated proteins which are targeted for degradation by the ubiquitin pathway by isolating ubiquitinated proteins that bound to the ubiquitin-specific-binding protein in step (c); and
(e) analyzing the isolated ubiquitinated proteins of step (d) which are targeted for degradation by the ubiquitin pathway by comparing pattern and abundance of said ubiquitinated proteins of step (d) from the disease or pathological biological sample with pattern and abundance of ubiquitinated proteins of a control sample to establish the expression profile of ubiquitinated proteins which are targeted for degradation by the ubiquitin pathway for the biological sample.
2. The method of claim 1 , further comprising release of the bound ubiquitinated proteins of step (d) from the affinity matrix by a de-ubiquitination reaction to generate isolated de-ubiquitinated proteins.
3. The method of claim 2 , wherein the analyzing step (e) is conducted by subjecting the isolated de-ubiquitinated proteins to high-resolution 2-dimensional (2D) gel electrophoresis.
4. The method of claim 3 , wherein 2D gel electrophoresis is followed by visualization of the isolated de-ubiquitinated proteins.
5. The method of claim 4 , wherein visualization is achieved with silver nitrate staining.
6. The method of claim 1 , further comprising release of the bound ubiquitinated proteins of step (d) from the affinity matrix by exposure to high salt conditions, detergent, or enzymatic reaction.
7. The method of claim 6 , wherein the bound ubiquitinated proteins of step (d) are released from the affinity matrix by exposure to ubiquitin-isopeptidase.