Bactericidal domain of lactoferrin: detection, quantitation, and characterization of lactoferricin in serum by SELDI affinity mass spectrometry.
Lactoferricin is a bioactive peptide fragment (3196 Da) derived from lactoferrin (80 kDa) that contains the bactericidal domain and the lymphocyte receptor-binding domain of lactoferrin. Although lactoferricin has been produced from lactoferrin by proteolytic digestion in vitro, its natural occurrence and distribution in vivo are still not clear, in part because of the absence of a suitable detection means. Surface-enhanced laser desorption/ionization (SELDI) was used to detect and characterize lactoferricin by affinity mass spectrometry. Human, porcine, and bovine lactoferricin in unfractionated serum samples were found to bind specifically to ligands presenting a terminal n-butyl group. SELDI was used to detect and quantify each species of lactoferricin in a manner that was independent of the presence of intact lactoferrin, partially degraded lactoferrin, and lactoferrin peptides containing the lactoferricin peptide sequence. The limit of detection of bovine lactofericin in serum was as low as 200 pg/ml. The FKCRRWQWR-homoserine/-homoserine lactone moiety of bovine lactoferricin, which includes the complete antimicrobial center (i.e., RRWQWR), was shown to be responsible for interaction with the n-butyl group. The SELDI procedure defined here is the only molecular recognition tool known to date that is capable of distinguishing the multi-functional lactoferricin domain located within structurally related but distinct forms of lactoferrin and its metabolic fragments. Enabling the direct quantitation of lactoferricin produced in vivo opens new opportunities to evaluate lactoferrin function.
Kuwata
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Department of Food Science and Technology, University of California, Davis 95616, USA. Hidi@msn.com
Yip
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Tomita
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Hutchens
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006801:Humans; D007781:Lactoferrin / Q000097:blood / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007781:Lactoferrin