An immuno-Matrix Assisted Laser Desorption/Ionization (iMALDI) method has been developed using anti-IgG beads to capture anti-AngI and anti-AngII antibodies, which are incubated with a ∼50μL plasma sample to which known amounts of stable-isotope-labeled AngI and AngII have been added. After a short incubation time, the beads are washed, placed directly on a MALDI target, and analyzed by mass spectrometry (MS) and tandem mass spectrometry (MS/MS). The iMALDI assay developed can detect and quantify angiotensin I (AngI) and angiotensin II (AngII) in human plasma. This assay has a Limit of Detection (LOD) of ∼10amol/μL (or ∼13pg/mL AngI and ∼11pg/mL AngII), at a S/N of 2:1, using only one-tenth of the antibody beads which were incubated with a 50-μL plasma sample. This LOD is within the relevant range of patient samples. Little or no angiotensin generation period is required, resulting in a rapid assay. Correlation coefficients for the standard curves are >0.99, with a linear range of 4-100fmol/μL (5-130ng/mL) and 100-2500amol/μL (106-2614pg/mL) for AngI and AngII, respectively. This duplexed assay can quantify AngI and AngII peptide levels simultaneously, in plasma from normotensive and hypertensive patients. The assay can detect changes in the levels of these peptides over time, which will allow quantitation of plasma renin and ACE activities.
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