In-Depth Characterization of N-Linked Oligosaccharides Using Fluoride-Mediated Negative Ion Microfluidic Chip LC鈥揗S
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- 作者：Wenqin Ni ; Jonathan Bones ; Barry L. Karger
- 刊名：Analytical Chemistry
- 出版年：2013
- 出版时间：March 19, 2013
- 年：2013
- 卷：85
- 期：6
- 页码：3127-3135
- 全文大小：594K
- 年卷期：v.85,no.6(March 19, 2013)
- ISSN：1520-6882
文摘
Characterization of N-glycans by liquid chromatography鈥損ositive electrospray ionization (ESI) tandem mass spectrometry (LC鈥揗S/MS) using a microfluidic chip packed with porous graphitized carbon (PGC) represents a rapidly developing area in oligosaccharide analysis. Positive ion ESI-MS generates B/Y-type glycosidic fragment ions under collisional-induced dissociation (CID). Although these ions facilitate glycan sequencing, they provide little information on linkage and positional isomers. Isomer identification in these cases is by retention on the PGC stationary phase where the specific structural isomers can, in principle, be separated. In this paper, we broaden the applicability of the PGC microfluidic chip/MS platform by implementing fluoride-mediated negative ESI-MS. Ammonium fluoride, added to the mobile phase, aids in the formation of pseudomolecular oligosaccharide anions due to the ability of fluoride to abstract a proton from the glycan structure. The negative charge results in the generation of C-type glycosidic fragments, highly informative A-type cross-ring fragment ions, and additional gas-phase ion reaction products (e.g., D- and E-type ions), which, when combined, lead to in-depth oligosaccharide characterization, including linkage and positional isomers. Due to the separation of anomers by the PGC phase, comparison of oligosaccharides with an intact reducing terminus to their experimentally prepared corresponding alditols was performed, revealing a more sensitive MS and, especially, MS/MS analysis from the glycans with a free reducing end. Fluoride also ensured recovery of charged oligosaccharides from the PGC stationary phase. Application to the characterization of N-glycans released from polyclonal human and murine serum IgG is presented to demonstrate the effectiveness of the chip/negative ESI approach.