Process to determine enzyme activity
View Patent ↗The present invention provides for various processes for determining enzyme activity. In one embodiment, a process for detecting an enzyme activity includes contacting a blood component with a substrate comprising a cleavage site of the enzyme and a tag moiety to produce a sample comprising a cleavage product having a known mass, and placing the sample in contact with a SELDI sample chip which has surface moieties which bind to the cleavage product. In another embodiment, the process further includes subjecting the SELDI sample chip to SELDI mass spectrometry to identify the cleavage product. The present invention also provides for a kit which includes a SELDI sample chip having surface moieties capable of binding to an enzyme cleavage product.
1. A process for detecting an enzyme activity, comprising:
preparing a sample by contacting a blood product with a substrate comprising a cleavage site of the enzyme and a tag moiety, wherein cleavage of the substrate by the enzyme produces a cleavage product having the tag moiety; and then
contacting the sample with a surface moiety that is disposed on a Surface Enhanced Laser Desorption/Ionization (SELDI) sample chip and that binds the tag moiety of the cleavage product, such that binding of the surface moiety to the cleavage product indicates activity of the enzyme in the blood product.
2. The process of claim 1 , further comprising, after contacting the sample with the surface moiety, subjecting the SELDI sample chip to SELDI mass spectrometry to identify the cleavage product.
3. The process of claim 2 , further comprising, after contacting the sample with the surface moiety and prior to subjecting the SELDI sample chip to SELDI mass spectometry,
washing the SELDI sample chip to remove blood product components that do not selectively bind to the surface moiety on the SELDI sample chip.
4. A process for detecting ADAMTS-13 activity, comprising:
preparing a sample by contacting a blood product with a substrate comprising an ADAMTS-13 cleavage site and a tag moiety, wherein cleavage of the substrate by ADAMTS-13 produces a cleavage product having a the tag moiety; and then
contacting sample with a surface moiety that is disposed on a SELDI sample chip and binds the tag moiety of the cleavage product, such that binding of the surface moiety to the cleavage product indicates ADAMTS-13 activity in the blood component.
5. The process of claim 4 , further comprising, after contacting the sample with the surface moiety,
washing the SELDI sample chip to remove blood product components that do not selectively bind to the surface moieties of the SELDI sample chip; and then
subjecting the SELDI sample chip to SELDI mass spectrometry to identify the cleavage product.
6. The process of claim 5 , further comprising quantifying the amount of cleavage product present in the sample.
7. The process of claim 4 , further comprising, prior to contacting the sample with the surface moiety,
terminating the cleavage and adding to the sample an internal control that binds the surface moiety.
8. The process of claim 7 , further comprising,
washing the SELDI sample chip after the internal control is added to the sample; and then
subjecting the SELDI sample chip to SELDI mass spectrometry to identify and determine the amount of the cleavage product and the internal control.
9. The process of claim 4 , wherein the tag moiety is a selected from the group consisting off a maltose binding protein tag, a glutathione-S-transferase (GST) tag, a biotin tag, an avidin tag and a histidine tag.
10. The process of claim 4 , wherein the tag moiety comprises a histidine tag.
11. The process of claim 4 , wherein the SELDI sample chip is charged with a compound comprising a metal selected from the group consisting of nickel, iron II, iron III, calcium, magnesium, manganese, copper, zinc and mixtures thereof.
12. The process of claim 4 , wherein the SELDI sample chip is an IMAC (immobilized metal affinity capture) ProteinChip.
13. The process of claim 4 , wherein the SELDI sample chip is charged with nickel sulfate.
14. The process of claim 4 , wherein the contacting of the blood product with the substrate is for a period which ranges from about 1 to about 20 hours.
15. A process for detecting ADAMTS-13 activity comprising:
preparing a sample by contacting a blood product with a substrate comprising an ADAMTS-13 cleavage site and a tag moiety, wherein cleavage of the substrate by ADAMTS-13 produces a cleavage product having the tag moiety; and
contacting the sample with a surface moiety that is disposed on a SELDI sample chip and binds the tag moiety of the cleavage product;
removing from the SELDI sample chip blood product components that do not selectively bind to the surface moieties moiety on the SELDI sample chip; and then
subjecting the SELDI sample chip to a SELDI mass spectrometer to identify the cleavage product, which indicates ADAMTS-13 activity.
16. The process of claim 15 , further comprising, prior to contacting the sample with the surface moiety,
terminating the cleavage and adding to the sample an internal control that binds the surface moiety.
17. The process of claim 15 , further comprising quantifying the amount of cleavage product present in the sample.
18. The process of claim 15 , wherein the amount of cleavage product present in the reaction sample is quantitatively compared to at least one internal control.
19. The process of claim 18 , wherein the amount of cleavage product is determined by the ratio of the amount of cleavage product measured by the mass spectrometer to the amount of the internal control measured by the mass spectrometer.
20. The process of claim 15 , wherein the tag moiety is a selected from the group consisting of a maltose binding protein tag, a glutathione-S-transferase (GST) tag, a biotin tag, an avidin tag and a histidine tag.
21. The process of claim 15 , wherein the SELDI sample chip is charged with a compound comprising a metal selected from the group consisting of nickel, iron II, iron III, calcium, magnesium, manganese, copper, zinc and mixtures thereof.
22. The process of claim 15 , wherein the tag moiety is a histidine tag and the SELDI sample chip is charged with a compound comprising nickel.
23. The process of claim 22 , wherein the SELDI sample chip is an IMAC (immobilized metal affinity capture) ProteinChip charged with nickel sulfate.
24. The process of claim 15 , wherein the contacting of the blood product with the substrate is for a period which ranges from about 1 to about 20 hours.
25. The process of claim 24 , wherein the period ranges from about 1 hours to about 2 hours to determine a level of ADAMTS-13 activity as low as 2.5%.
26. The process of claim 24 , wherein the incubation period ranges from about 10 hours to about 40 hours to determine a level of ADAMTS-13 activity as low as 0.31%.
27. The process of claim 15 , wherein the substrate is purified.
28. The process of claim 15 , wherein the substrate is engineered.
29. The process of claim 15 , wherein the substrate comprises a recombinant protein with a tag moiety that produces a cleavage product upon proteolysis.
30. The process of claim 15 , wherein the SELDI mass spectrometer is a SELDI-TOS mass spectrometer.
31. The process of claim 15 , wherein the process detects the ability of an ADAMTS-13 antibody inhibitor from a patient to neutralize ADAMTS-13 activity in normal plasma.
32. The process of claim 15 , wherein the amount of the cleavage product is used to diagnose thrombotic thrombocytopenic purpura (TTP).
33. The process of claim 15 , wherein the amount of the cleavage product is used to diagnose blood clotting disorders.
34. The process of claim 33 , wherein the blood clotting disorder is selected from the group consisting of heart attack, stroke, deep vein thrombosis and arterial thrombodic disorders.
35. The process of claim 15 , wherein the amount of the cleavage product is used to assess a response to therapy.
36. The process of claim 15 , wherein the amount of the cleavage product is used to predict recurrence of TTP.