Estimation of angiotensin peptides in biological samples by LC–MS method

Abstract

The low abundance of angiotensin peptides in biological tissues such as the kidney cortex, adipose tissue, urine and plasma makes their detection and quantification a challenge. A few available methods used to quantify these peptides involve lengthy processes of sample preparation and are hardly quantitative. Here, we report a mass spectrometry approach for quantifying angiotensin peptides [Ang II, Ang-(1-7)] in the kidney cortex, epididymal white adipose tissue (eWAT), urine and plasma of male mice. Tissue homogenates, urine and plasma samples were solid-phase extracted with C18 Sep-Pak cartridges and eluted off proteinaceous compounds. These extracted peptide samples were separated on C18 column with a linear acetonitrile gradient and detected by Q-ToF mass analyzer in ESI+-MS ion mode based on their retention time, accurate mass measurement of peptides, the isotope pattern of doubly charged molecular ion, and quantitation of peak area (or ion count) when referencing to the angiotensin peptide standards. The lower limit of quantitation for each angiotensin peptide was 10 pgmg−1 with the percent recovery at 100.6%. The intra-batch precision for Ang-(1-7) and Ang II were 24.0 and 12.7%, accuracy 84.0–123.0% and 100.2–116.0% respectively. Using this method, we determined the levels of Ang II and Ang-(1-7) in the kidney cortex, eWAT, urine and plasma. Quantification of angiotensin peptides could help target subtle therapeutics changes against pathophysiological conditions such as obesity, kidney disease and hypertension.

Biochemical evaluation of the renin-angiotensin system: the good, bad, and absolute?

“…Finally, Ali et al (7) quantified plasma ANG II and ANG-(1-7) content at 20,000 and 40,000 pM, respectively, in WT mice using UPLC-quardruple (Q)TOF. The presence of other ANG II or ANG-(1-7) metabolites were not reported, but the extreme ANG II and ANG-(1-7) values by this method likely reflect inadequate inhibition of plasma peptidases during blood collection and extraction (7).…”

“…The ANG II metabolite ANG- (3)(4)(5)(6)(7)(8) or ANG IV has distinct functional properties through the interaction with the insulin-regulated aminopeptidase, although the peptide also stimulates the AT 1 R (4, 80, 150). ANG-(1-9), a potential product of ACE2 processing of ANG I, appears to functionally interact with the AT 2 receptor (101).…”

“…Danser and colleagues (113,139) suggest that urinary renin may be an alternative measure of an active RAS within the kidney. Quenched fluorescent substrates based on the ANG- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) sequence are available to measure renin. Although this approach does not rely on the detection of ANG I or an exogenous source of angiotensinogen, the sensitivity and specificity of the peptide substrates are not comparable with angiotensinogen.…”

“…Conversely, alternative RAS pathways may mitigate against the activation of the ACE-ANG II-AT 1 R, particularly in response to the pharmacological blockade of this axis (29,43,47,119). Targeting ACE reduces ANG II expression but enhances the ANG-(1-7)-AT 7 /MasR axis that generally opposes the actions of the ANG II-AT 1 R (29,43,44,103,119,120). AT 1 R antagonists may also increase the formation of ANG-(1-7) through ACE2, as well as shunt ANG II to the AT 2 R pathway that shares similar properties to the ANG-(1-7) system (12,25,29,44,47,119).…”

Development of a High-Sensitivity Quantitation Method for Arginine Vasopressin by High-Performance Liquid Chromatography Tandem Mass Spectrometry, and Comparison with Quantitative Values by Radioimmunoassay

“…The use of a 0.1% formic acid aqueous solution and acetonitrile improved the detection sensitivity of peptide quantitation. [23][24][25][26][27] However, in this work, our comparison revealed that methanol was better than acetonitrile in terms of sensitivity, and that a low acetic acid concentration (0.01%) was preferable over the use of formic acid. In general, acetonitrile is used in ESI ionization, because an organic solvent with low viscosity and a low boiling point is preferred for ionization.…”