Detecting low abundance vasoactive peptides in plasma; progress towards absolute quantitation using nano LC-MS

摘要

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 utilizing nano LC-MS to simultaneously measure a select group of vasoactive peptides (angiotensin, bradykinin and related hormones) in 50 μl 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 offline 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.