| Autor: |
Code C; Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark.; Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark., Qiu D; Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark., Solov'yov IA; Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.; Department of Physics, Carl von Ossietzky University Oldenburg, Carl-von-Ossietzky-Str. 9-11, 26129 Oldenburg, Germany.; Research Centre for Neurosensory Science, Carl von Ossietzky Universität Oldenburg, Carl-von-Ossietzky-Str. 9-11, 26129 Oldenburg, Germany.; Center for Nanoscale Dynamics (CENAD), Carl von Ossietzky Universität Oldenburg Institut für Physik, Ammerländer Heerstr. 114-118, 26129 Oldenburg, Germany., Lee JG; Center for Molecular Intelligence, The State University of New York (SUNY), Korea, 119 Songdo Munwha-ro, Yeonsu-gu, 21985 Incheon, Korea., Shin HC; Center for Molecular Intelligence, The State University of New York (SUNY), Korea, 119 Songdo Munwha-ro, Yeonsu-gu, 21985 Incheon, Korea., Roland C; Department of Physics, North Carolina State University, Raleigh, North Carolina 27695, United States., Sagui C; Department of Physics, North Carolina State University, Raleigh, North Carolina 27695, United States., Houde D; Department of Protein Pharmaceutical Development, Biogen, Cambridge, Massachusetts 02142, United States., Rand KD; Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen, Denmark., Jørgensen TJD; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark. |
| Abstrakt: |
Protein glycosylation is a common post-translational modification on extracellular proteins. The conformational dynamics of several glycoproteins have been characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS). However, it is, in most cases, not possible to extract information about glycan conformation and dynamics due to the general difficulty of separating the deuterium content of the glycan from that of the peptide (in particular, for O-linked glycans). Here, we investigate whether the fragmentation of protonated glycopeptides by collision-induced dissociation (CID) can be used to determine the solution-specific deuterium content of the glycan. Central to this concept is that glycopeptides can undergo a facile loss of glycans upon CID, thereby allowing for the determination of their masses. However, an essential prerequisite is that hydrogen and deuterium (H/D) scrambling can be kept in check. Therefore, we have measured the degree of scrambling upon glycosidic bond cleavage in glycopeptides that differ in the conformational flexibility of their backbone and glycosylation pattern. Our results show that complete scrambling precedes the glycosidic bond cleavage in normal glycopeptides derived from a glycoprotein; i.e., all labile hydrogens have undergone positional randomization prior to loss of the glycan. In contrast, the glycosidic bond cleavage occurs without any scrambling in the glycopeptide antibiotic vancomycin, reflecting that the glycan cannot interact with the peptide moiety due to a conformationally restricted backbone as revealed by molecular dynamics simulations. Scrambling is also inhibited, albeit to a lesser degree, in the conformationally restricted glycopeptides ristocetin and its pseudoaglycone, demonstrating that scrambling depends on an intricate interplay between the flexibility and proximity of the glycan and the peptide backbone. |
