Quantification of angiotensin-converting-enzyme-mediated degradation of human chemerin 145-154 in plasma by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.
Chemerin is a chemoattractive protein acting as a ligand for the G-protein-coupled receptor ChemR23/CMKLR1 and plays an important role in the innate and adaptive immunity. Proteolytic processing of its C terminus is essential for receptor binding and physiological activity. Therefore, we investigated the plasma stability of the decapeptide chemerin 145-154 (P(145)-F(154)) corresponding to the C terminus of the physiologically active chemerin variant E(21)-F(154) from human hemofiltrate. For monitoring concentration-time profiles and degradation products we developed a novel matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry procedure using an internal peptide standard (hemorphin LVV-H7) for quantification. The linear range covers 2.5 orders of magnitude in the lower micromolar concentration range (lower limit of quantification 0.312 microg/ml, 0.25 microM) characterized by satisfactory reproducibility (CV < or =9%), accuracy (< or =10%), ruggedness, and recovery (98%). We found that chemerin 145-154 is C-terminally truncated in human citrate plasma by the cleavage of the penultimate dipeptidyl residue. N-terminal truncation was not observed. In contrast to citrate plasma, no degradation was detected in ethylenediammetetraacetate (EDTA) plasma. We identified angiotensin-converting-enzyme (ACE) to be responsible for C-terminal truncation, which could be completely inhibited by EDTA and captopril. These results are relevant to clarify the natural processing of chemerin and the potential involvement of ACE in mediating the immune response.
John
Harald
H
IPF PharmaCeuticals GmbH, Feodor-Lynen-Str. 31, D-30625 Hannover, Germany. HaraldJohn@bundeswehr.org
Hierer
Jessica
J
Haas
Olga
O
Forssmann
Wolf-Georg
WG
D000595:Amino Acid Sequence; D018925:Chemokines / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D018925:Chemokines; D036341:Intercellular Signaling Peptides and Proteins; C478630:RARRES2 protein, human; D007703:Peptidyl-Dipeptidase A