title: Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling.
abstract: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for high-throughput glycan profiling analysis. In spite of the biological importance of sialic acids on nonreducing ends of glycans, it is still difficult to analyze glycans containing sialic acid residues due to their instability and the presence of linkage isomers. In this Article, we describe a one-pot glycan purification/derivatization method employing a newly developed linkage-specific sialic acid derivatization for MS-based glycan profiling with differentiation of sialyl linkage isomer. The derivatization, termed sialic acid linkage specific alkylamidation (SALSA), consists of sequential two-step alkylamidations. As a result of the reactions, α2,6- and α2,3-linked sialic acids are selectively amidated with different length of alkyl chains, allowing distinction of α2,3-/α2,6-linkage isomers from given mass spectra. Our studies using N-glycan standards with known sialyl linkages proved high suitability of SALSA for reliable relative quantification of α2,3-/α2,6-linked sialic acids compared with existing sialic acid derivatization approaches. SALSA fully stabilizes both α2,3- and α2,6-linked sialic acids by alkylamidation; thereby, it became possible to combine SALSA with existing glycan analysis/preparation methods as follows. The combination of SALSA and chemoselective glycan purification using hydrazide beads allows easy one-pot purification of glycans from complex biological samples, together with linkage-specific sialic acid stabilization. Moreover, SALSA-derivatized glycans can be labeled via reductive amination without causing byproducts such as amide decomposition. This solid-phase SALSA followed by glycan labeling has been successfully applied to human plasma N-glycome profiling.
authors.affiliation: Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation , 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan.
authors.forename: Takashi
authors.identifier: 0000-0002-3537-8316
authors.initials: T
authors.lastname: Nishikaze
authors.affiliation: Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology , 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tsumoto
authors.affiliation: Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation , 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan.
authors.forename: Sadanori
authors.initials: S
authors.lastname: Sekiya
authors.affiliation: Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation , 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Iwamoto
authors.affiliation: Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology , 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Miura
authors.affiliation: Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation , 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Tanaka