A Multiplexed, and Quantitative immuno-MALDI (iMALDI) Assay for Diagnosing Secondary Hypertension

摘要

By direct MALDI MS analysis, we were unable to detect endogenous angiotensin I or II in normal hypertensive human plasma. However, using the iMALDI approach we successfully detected both endogenous angiotensin I and angiotensin II in human plasma. The sequences of angiotensin I and II were confirmed by tandem mass spectrometry. We have also previously shown detection of angiotensin I from whole human plasma by iMALDI without incorporating an angiotensin I generation period. This direct assay offers advantages in time and specificity compared to plasma renin activity assays for diagnosing and monitoring hypertension. Our iMALDI approach also provides an improved limit of detection of synthetic angiotensin I in buffer by 80-fold compared to angiotensin I spotted directly on a MALDI target. The limit of detection for angiotensin I was determined to be 65 pg/mL, thus meeting the clinical requirements for measuring low renin activity. By modifying the iMALDI approach for a multiplexed assay, we have demonstrated simultaneous detection of angiotensin I and II in human plasma. The concentration curves show that the assay is linear from 0 fmol/uL to 100 fmol/uL and 0 amol/uL to 2500 amol/uL with correlation coefficients of 0.99 for angiotensin I and angiotensin II respectively. Simultaneous detection of angiotensin I and II is also advantageous for assessing plasma renin and ACE activity, and provides the ability to comprehensively assess the system's ability to regulate arterial pressure, thus providing stronger insight towards disease diagnosis and treatment.