title: Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy.
abstract: A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic16O/18O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol-1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per μl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease.
authors.affiliation: Department of Chemistry, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung 402, Taiwan.
authors.forename: Hsing-Fen
authors.initials: HF
authors.lastname: Tsai
authors.affiliation: Department of Chemistry, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung 402, Taiwan. Electronic address: hhhsiao@dragon.nchu.edu.tw.
authors.forename: He-Hsuan
authors.initials: HH
authors.lastname: Hsiao
mesh_terms: D006509:Hepatitis B / Q000175:diagnosis*; D006514:Hepatitis B Surface Antigens / Q000737:chemistry; D006513:Hepatitis B e Antigens / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling; D057230:Limit of Detection; D013058:Mass Spectrometry; D010103:Oxygen Isotopes; D010455:Peptides / Q000138:chemical synthesis*; D040901:Proteomics; D014357:Trypsin
chemical_list: D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D010103:Oxygen Isotopes; D010455:Peptides; D014357:Trypsin
keywords: Filter-assisted enzymatic labeling; Hepatitis B virus; Mass spectrometry; Parallel reaction monitoring; Stable isotopically labeled peptides