title: Glycopeptide identification using liquid-chromatography-compatible hot electron capture dissociation in a radio-frequency-quadrupole ion trap.
abstract: We developed a liquid chromatography (LC) compatible electron capture dissociation (ECD) mass spectrometer for glycoproteomics, with which ECD and hot ECD (HECD) experiments can be flexibly switched by quickly changing the electron energy without further tuning of the mass spectrometer. Desialylated glycopeptides were dissociated well in both ECD and HECD experiments. For sialylated glycopeptides, on the other hand, ECD with electron energy higher than 4 eV showed significantly higher sequence coverage than that with an electron energy of 0.2 eV. A nano LC system was coupled to our ECD mass spectrometer to investigate N-linked glycopeptides from lysylendopeptidase (Lys-C) digests of human transferrin. ECD spectra at multiple electron energies of 0.2, 5.0, and 9.0 eV were obtained for each targeting precursor ion in a single LC injection. Glycopeptides with a sialylated bi-, tri-, or tetra-antennary complex N-glycan were identified with high sequence coverage by HECD. Glycopeptides with tri- or tetra-antennary N-glycans have seldom been analyzed by ECD or ETD before this report. We also found that a preferential dissociation of nonreducing termini of glycans in glycopeptides by ECD and HECD.
authors: Central Research Laboratory, Hitachi, Ltd., 1-280 Higashi-Koigakubo, Kokubunji, Tokyo 185-8601, Japan.
authors: Naomi
authors: N
authors: Manri
authors: Hiroyuki
authors: H
authors: Satake
authors: Akihito
authors: A
authors: Kaneko
authors: Atsumu
authors: A
authors: Hirabayashi
authors: Takashi
authors: T
authors: Baba
authors: Takeshi
authors: T
authors: Sakamoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004527:Egg Proteins / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D007477:Ions / Q000737:chemistry; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry; D061250:Transferrins / Q000378:metabolism
chemical_list: D004527:Egg Proteins; D006020:Glycopeptides; D007477:Ions; D061250:Transferrins; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase