Identification of incompletely processed potential carboxypeptidase E substrates from CpEfat/CpEfat mice.
In an attempt to identify peptides that may be involved in the obese phenotype observed in CpEfat/CpEfat mice (deficient in Carboxypeptidase E, CpE) samples from fourteen neuroendocrine tissues in wild-type and CpEfat/CpEfat mice were obtained. Peptides were purified from these tissues and potential CpE substrate peptides were enriched using an anhydrotrypsin column that captures peptides with basic C-termini. Bound peptides were subjected to tryptic digestion and followed by liquid chromatography-mass spectrometry analysis. The relative levels of CpEfat/CpEfat versus wild-type peptides were determined by comparison of the ion intensities. Peptide ions elevated in the CpEfat/CpEfat samples were identified by targeted liquid chromatography-tandem mass spectrometry. From those ions, 27 peptides derived from known neuropeptides (including CpE substrates) were identified, together with another 25 peptides from proteins not known to be components of the neuropeptide processing pathway. The known CpE substrates identified included the recently discovered proSAAS, granin-like neuroendocrine peptide precursor that inhibits prohormone processing. The approach demonstrated the feasibility of using an affinity-based method for identifying differences in specific classes of peptides between normal and mutant mice.
Bures
E J
EJ
Departments of Biochemistry and Genetics, Amgen, Thousand Oaks, CA, USA.
Courchesne
P L
PL
Douglass
J
J
Chen
K
K
Davis
M T
MT
Jones
M D
MD
McGinley
M D
MD
Robinson
J H
JH
Spahr
C S
CS
Sun
J
J
Wahl
R C
RC
Patterson
S D
SD
D000595:Amino Acid Sequence; D000818:Animals; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000235:genetics* / Q000378:metabolism*; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D009490:Neurosecretory Systems / Q000378:metabolism; D009765:Obesity / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome; D013379:Substrate Specificity; D014018:Tissue Distribution
D009479:Neuropeptides; D011498:Protein Precursors; D020543:Proteome; D002268:Carboxypeptidases; D043423:Carboxypeptidase H