In vivo multiplex quantitative analysis of 3 forms of alpha melanocyte stimulating hormone in pituitary of prolyl endopeptidase deficient mice

Abstract

Background: In vitro reactions are useful to identify putative enzyme substrates, but in vivo validation is required to identify actual enzyme substrates that have biological meaning. To investigate in vivo effects of prolyl endopeptidase (PREP), a serine protease, on alpha melanocyte stimulating hormone (alpha-MSH), we developed a new mass spectrometry based technique to quantitate, in multiplex, the various forms of alpha-MSH.

Methods: Using Multiple Reaction Monitoring (MRM), we analyzed peptide transitions to quantify three different forms of alpha-MSH. Transitions were first confirmed using standard peptides. Samples were then analyzed by mass spectrometry using a triple quadrupole mass spectrometer, after elution from a reverse phase C18 column by a gradient of acetonitrile.

Results: We first demonstrate in vitro that PREP digests biological active alpha melanocyte stimulating hormone (alpha-MSH(1-13)), by cleaving the terminal amidated valine and releasing a truncated alpha melanocyte stimulating hormone (alpha-MSH(1-12)) product--the 12 residues alpha-MSH form. We then use the technique in vivo to analyze the MRM transitions of the three different forms of alpha-MSH: the deacetylated alpha-MSH(1-13), the acetylated alpha-MSH(1-13) and the truncated form alpha-MSH(1-12). For this experiment, we used a mouse model (PREP-GT) in which the serine protease, prolyl endopeptidase, is deficient due to a genetrap insertion. Here we report that the ratio between acetylated alpha-MSH(1-13) and alpha-MSH(1-12) is significantly increased (P-value = 0.015, N = 6) in the pituitaries of PREP-GT mice when compared to wild type littermates. In addition no significant changes were revealed in the relative level of alpha-MSH(1-13) versus the deacetylated alpha-MSH(1-13). These results combined with the demonstration that PREP digests alpha-MSH(1-13) in vitro, strongly suggest that alpha-MSH(1-13) is an in vivo substrate of PREP.

Conclusion: The multiplex targeted quantitative peptidomics technique we present in this study will be decidedly useful to monitor several neuropeptide enzymatic reactions in vivo under varying conditions.

Figure 1

The seven α-MSH_1–12 MRM transitions (MRMT_1 to MRMT_7) used are increasing in a synchronous fashion during the time course. Peak areas of each MRM transition are shown for each time point on a log scale.

Figure 2

In vitro time course of PREP activity on α-MSH_1–13. The increase of α-MSH_1–12 (pink line) fits a linear regression with R² = 0.9370 (black line). The ratio of α-MSH_1–13/α-MSH_1–12 decreases in a corresponding fashion (blue line).

Figure 3

Western blots of pituitary from PREP-GT littermate mice with PREP antibody: From left to right Wild Type (WT), PREP-GT heterozygotes (HT) and PREP-GT homozygotes (GT). The loading is shown below from a Ponceau S stained gel.

Figure 4

ratio of acetylated α-MSH_1–13 acetylated over deacetylated α-MSH_1–13 and acetylated α-MSH_1–13 over α-MSH_1–12 in three genotypes of littermate mice from PREP-GT mouse strain. Left: acetylated α-MSH_1–13/deacetylated α-MSH_1–13; Right: α-MSH_1–13/α-MSH_1–12.