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

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 (α-MSH), we developed a new mass spectrometry based technique to quantitate, in multiplex, the various forms of α-MSH. METHODS: Using Multiple Reaction Monitoring (MRM), we analyzed peptide transitions to quantify three different forms of α-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 (α-MSH(1–13)), by cleaving the terminal amidated valine and releasing a truncated alpha melanocyte stimulating hormone (α-MSH(1–12)) product – the 12 residues α-MSH form. We then use the technique in vivo to analyze the MRM transitions of the three different forms of α-MSH: the deacetylated α-MSH(1–13), the acetylated α-MSH(1–13 )and the truncated form α-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 α-MSH(1–13 )and α-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 α-MSH(1–13 )versus the deacetylated α-MSH(1–13). These results combined with the demonstration that PREP digests α-MSH(1–13) in vitro, strongly suggest that α-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.