New mass spectrometric assay for angiotensin-converting enzyme 2 activity.
A novel assay was developed for evaluation of mouse angiotensin-converting enzyme (ACE) 2 and recombinant human ACE2 (rACE2) activity. Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (MS) with ProteinChip Array technology, ACE1 and ACE2 activity could be measured using natural peptide substrates. Plasma from C57BL/6 mice, kidney from wild-type and ACE2 knockout mice, and rACE2 were used for assay validation. Plasma or tissue extracts were incubated with angiotensin I (Ang I; 1296 m/z) or angiotensin II (Ang II; 1045 m/z). Reaction mixtures were spotted onto the ProteinChips WCX2 and peptides detected using surface-enhanced laser desorption/ionization time of flight MS. MS peaks for the substrates, Ang I and Ang II, and the generated peptides, Ang (1-7) and Ang (1-9), were monitored. The ACE2 inhibitor MLN 4760 (0.01 to 100 micromol/L) significantly inhibited rACE2 activity (IC50=3 nmol/L). Ang II was preferably cleaved by rACE2 (km=5 mumol/L), whereas Ang I was not a good substrate for rACE2. There was no detectable ACE2 activity in plasma. Assay specificity was validated in a model of ACE2 gene deletion. In kidney extract from ACE2-deficient mice, there was no generation of Ang (1-7) from Ang II. However, Ang (1-7) was produced when Ang I was used as a substrate. In conclusion, we developed a specific and sensitive assay for ACE2 activity, which used the natural endogenous peptide substrate Ang II. This approach allows for the rapid screening for ACE2, which has applications in drug testing, high-throughput enzymatic assays, and identification of novel substrates/inhibitors of the renin-angiotensin system.
Elased
Khalid M
KM
Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA. Khalid.elased@wright.edu
Cunha
Tatiana S
TS
Gurley
Susan B
SB
Coffman
Thomas M
TM
Morris
Mariana
M
D000803:Angiotensin I / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D000804:Angiotensin II / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000097:blood / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis; D007703:Peptidyl-Dipeptidase A / Q000032:analysis* / Q000097:blood / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000097:blood; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; C506331:angiotensin I (1-9); D000804:Angiotensin II; D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A; C118790:angiotensin I (1-7)