PYY(1-36) is the major form of PYY in rat distal small intestine: Quantification using high-resolution mass spectrometry

We measured molecular forms of PYY in the distal half of rat small intestine using a new method for tissue extraction, three sequential reverse phase chromatography steps, and PYY radioimmunoassay and mass spectrometry to measure their levels. The extraction method called RAPID, developed to minimize artifactual degradation of PYY during tissue extraction and sample preparation, uses Reduced temperature, Acidified buffer, Peptidase inhibitors, Isotopically enriched mass spectrometry standards, and Dilution to inhibit and monitor endogenous peptide degradation during tissue processing. Synthetic peptides [PYY(1-36)-NH2, PYY(3-36)-NH2, PYY(1-36)-Gly-OH, and PYY(3-36)-Gly-OH] selectively enriched with 13C3-alanine were added as internal standards to the extraction buffer. By collecting mass spectra rather than multiple-reaction-monitoring (MRM) profiles, we simultaneously screen for any PYY forms that were present in the immunoreactive fractions. PYY(1-36)-NH2, PYY(3-36)-NH2, PYY(1-36)-Gly-OH, and PYY(3-36)-Gly-OH were identified and quantified at 64.3 ± 4.5, 6.1 ± 0.9, 0.9 ± 0.1, and < 0.3 pmol/g of tissue, respectively (n = 3). Thus, we found that in rat distal small intestine proPYY is processed to PYY(1-36)-NH2 with little conversion to PYY(3-36)-NH2. These data suggest that production of PYY(3-36)-NH2 (a form with greater potency than PYY(1-36)-NH2 for inhibition of feeding and gastric emptying) occurs after the peptide leaves its cell of synthesis by enzymatic action in the circulation.