Screening assay for enzymes involved in pathophysiology using SELDI-TOF mass spectrometry
The invention provides methods for quantifying enzymatic activity of an enzyme with a known substrate. The methods employ SELDI-TOF mass spectrometry, and are suitable, in particular, for assaying aspects of the renin-angiotensin system. The methods may be utilized to assess and/or monitor biological conditions associated with the renin-angiotensin system prior to the manifestation of known physiological and biomarkers for such conditions. The methods are suitable for analysis of pharmacological effectors of the renin-angiotensin system, and are particularly suitable for automation and high-throughput screening assay design.
1. A method of quantifying the enzymatic activity of at least one enzyme selected from the group consisting of renin, ACE1 and ACE2 with a known substrate, the method comprising:
a) obtaining a biological sample comprising said at least one enzyme;
b) adding at least one known substrate of said at least one enzyme to the biological sample and incubating to generate enzymatic activity products in a mixture;
c) loading the mixture onto a protein chip to produce a loaded chip;
d) washing the loaded chip to remove any unbound proteins and contaminants;
e) reading the loaded chip using SELDI-TOF mass spectrometry;
f) generating spectral data using commercially available software compatible for this purpose;
g) analyzing the spectral data to identify the enzymatic activity products; and
h) subjecting the spectral data to an algorithm that permits quantification of the enzymatic activity products.
2. The method according to claim 1 , wherein the algorithm comprises an area-under-the-curve and/or peak height intensity calculations.
3. The method according to claim 1 , wherein the known substrate comprises angiotensinogen, angiotensin I, angiotensin II, or Ang(1-7).
4. The method according to claim 1 , wherein said at least one enzyme is ACE1 or ACE2.
5. The method according to claim 1 , wherein the enzymatic activity products comprise angiotensin I or angiotensin II.
6. The method according to claim 5 , wherein the enzymatic activity products comprises angiotensin II.
7. The method according to claim 1 , wherein the at least one enzyme comprises more than one enzyme and quantification is achieved in parallel for each of the more than one enzymes.
8. A method for determining whether an individual has an increased risk of developing diseases associated with the Renin-Angiotensin System, the method comprising: quantifying angiotensin II according to the method recited in claim 5 and comparing the quantification of the angiotension II levels to a standard.
9. The method of claim 1 , wherein said substrate is not labeled.
10. The method of claim 1 , wherein said biological sample has a volume of 0.5 to 2.5 μl or less.
11. The method of claim 1 , wherein the peak height ratio of products to substrate is measured.
12. The method of claim 1 , wherein said substrate and said biological sample are incubated in the presence of PMSF.