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Anal Biochem. 2002 Jul 1;306(1):124-34.

Synthesis and characterization of a collagen model delta-O-phosphohydroxylysine-containing peptide.

Author information

1
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

Abstract

The existence of delta-O-phosphohydroxylysine (HylP) residues in collagen has been reported. However, such phosphorylated residues have not been isolated nor has their location within the protein sequence been identified. To develop the analytical chemistry necessary for the identification of HylP in proteins, a model HylP-containing peptide, Phe-dl-HylP-Gly-Gln-Pro-Ala-Ile-Gly-Phe (I), was synthesized. The peptide was assembled using 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase synthesis; N(alpha)-Fmoc-dl-hydroxy-dl-Lys(N(epsilon)-tert-butyloxycarbonyl)-OH was used to incorporate Hyl, and global phosphitylation/oxidation was used to introduce the phosphate group. The pK(a2) of the phosphate group, as determined using 31P NMR, was 5.6. Phosphoamino acid analysis of I was performed using either dabsyl chloride or phenylisothiocyanate derivatization followed by microbore reversed-phase HPLC separation of N(alpha, epsilon)-didabsyl-HylP or N(alpha, epsilon)-diphenylthiocarbamyl-HylP. HylP was found to be relatively resistant to acid hydrolysis, allowing for its quantitation. Solid-phase Edman degradation of I was used for positive identification of N(alpha)-phenylthiohydantoin-N(epsilon)-phenylthiocarbamyl-HylP. The masses of the phenylthiohydantoin and dabsyl derivatives of HylP were confirmed by electrospray ionization triple-quadrupole (ESI-MS). Peptide I was positively identified as a phosphopeptide by ESI-MS/precursor-ion scanning. Low-energy ESI-MS/MS confirmed the position of HylP within the sequence of I. Phosphorylation of Hyl led to complete resistance of I to lysine-specific endopeptidases.

PMID:
12069423
DOI:
10.1006/abio.2002.5693
[Indexed for MEDLINE]

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