Detecting low-abundance vasoactive peptides in plasma: progress toward absolute quantitation using nano liquid chromatography-mass spectrometry

Abstract

Profiling changes in the concentration of functionally related peptide hormones is critical to understanding the etiology of many diseases and therapies. We present novel data using nano liquid chromatography-mass spectrometry (LC-MS) to simultaneously measure a select group of vasoactive peptides (angiotensin, bradykinin, and related hormones) in 50-microl plasma samples, enabling repeated sampling in rodent models. By chromatographically resolving target peptides and using multiple reaction monitoring to enhance MS sensitivity, linear responses down to 10(-17) mol were achieved. Purification of plasma peptides by either methanol precipitation or off-line high-performance liquid chromatography (HPLC) fractionation enabled the detection of endogenous peptides and revealed approaches for enhancing recovery. As proof of principle, seven vasoactive peptides were profiled before, during, and after acute angiotensin-converting enzyme (ACE) inhibition in an anesthetized rat. Of note was an apparent 10-fold increase in vasodilatory bradykinin that reversed after drug infusion but relatively minor changes in angiotensin II levels. Targeted MS analysis used to profile functionally related peptides or other analytes will greatly enhance our ability to define the sequence of events regulating complex and dynamic physiological processes.

Figure 1. Vasoactive peptides are substrates for ACE activity

Multiple cleavage products of both angiotensin I and bradykinin are known to regulate vascular tone, acting as ligands for specific receptors. Peptide processing by angiotensin converting enzymes (ACE) is a target for anti-hypertensive pharmacotherapy aiming to shift the concentration of these vasoactive peptides. Multiple steps in the synthesis and/or degradation of each peptide family are blocked by ACE inhibition (ACEi) but little is known regarding the relative changes in concentration among the various hormones.

Figure 2. Enhanced detection limits of LC-MS

Multiple reaction monitoring (MRM) increases sensitivity by detecting only the fragments derived from a specified parent ion. The MS² chromatogram depicted here shows the signal intensity of a fragment (m/z 534.3) derived from the main parent ion of vasopressin (ADH: m/z 543.0). The signal intensity from a predominant fragment may be extracted and used for quantification. The log × log plot insert of regression analysis shows linearity of response over 4 orders of magnitude down to 10⁻¹⁷ mol injected on column.

Figure 3. Elution and detection of 5 vasoactive peptides

Components of a freshly prepared standard (1.0 fmol injected on column) are resolved by nano-LC to enable optimal MRM analysis. Vertical divisions indicate programmed changes in detector settings, synchronized to appropriate elution times.

Figure 4. Detection of endogenous peptide hormones in rat plasma

Two chromatograms resulting from the analyses of plasma extracts with methanol (=5 μl plasma with and without the addition of 0.1 fmol standards; solid and hatched lines respectively). The table summarizes mean values from duplicate runs of each sample and the difference (Δ) resulting from addition of standards. Note that the spiked standard did not contain bradykinin 1-8.

Figure 5. Sample depletion due to nonspecific adsorption

a) A comparison of the linearity in dose response measurements with equimolar injections from stock solutions of differing concentration ([10⁻¹⁰ and 10⁻⁹M]). The accuracy and reproducibility of measurement is confirmed by multiple linear responses in either sample but smaller slopes produced by the [10⁻¹⁰M] indicate significant depletion. b) Processing a [10⁻⁹M] standard mix by off-line reverse phase HPLC fractionation and reconstitution with eluent “A” increased recovery.

Figure 6. Acute cardiovascular effect of ACEi

Administration of an angiotensin converting enzyme inhibitor (Captopril, 100 mg/kg/min IV) caused a rapid decrease in blood pressure and a reflex increase in heart rate within 15 min.

Figure 7. Vasoactive peptides in plasma before, during and after ACEi infusion

The relative signal intensity for 7 structurally and/or functionally related hormones was determined in an anesthetized rat. Blood samples (<100 μl) were drawn at 15 min intervals. Endogenous peptides were concentrated 10 fold and 2 μl was injected on column. Changes over time for each peptide are expressed as a percentage of control in the table.