Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling
Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling
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DOI：
10.1021/acs.analchem.6b04150
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发表时间：
2017-02-21
影响因子：
7.4
通讯作者：
Tanaka, Koichi
中科院分区：
文献类型：
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作者：
Nishikaze, Takashi;Tsurnoto, Hiroki;Tanaka, Koichi
基质辅助激光解吸电离质谱仪(MALDI-MS)已被用于高通量多糖图谱分析。尽管唾液酸在多糖的非还原末端具有重要的生物学意义，但由于唾液酸残基的不稳定性和键合异构体的存在，分析含唾液酸残基的多糖仍然是困难的。在这篇文章中，我们描述了一种一锅法的葡聚糖纯化/衍生化方法，该方法采用新开发的连接特异性唾液酸衍生化方法，用于区分唾液酸键合异构体的MS多糖图谱。该衍生反应称为唾液酸键特异性烷基酰胺化反应(SASA)，由两步烷基酰胺化反应组成。作为反应的结果，α2，6-和α2，3-连接的唾液酸被不同长度的烷基链选择性地酰胺化，从而从给定的质谱图中区分出α2，3-/α2，6-连接的异构体。我们使用已知唾液酸键的N-葡聚糖标准品的研究证明，与现有的唾液酸衍生化方法相比，SASA对于α2，3-/α2，6-连接唾液酸的可靠相对定量具有很高的适用性。SASA通过烷基酰胺化完全稳定α-2，3-和α-2，6-连接的唾液酸；因此，可以将SASA与现有的多糖分析/制备方法结合如下。SALSA和使用酰肼微珠的化学选择性多糖纯化相结合，使得从复杂的生物样品中轻松地一锅纯化多糖，以及连接特定的唾液酸稳定。此外，Salsa衍生的葡聚糖可以通过还原胺化反应进行标记，而不会产生酰胺分解等副产物。这种固相SASA结合葡聚糖标记已成功地应用于人血浆N-糖蛋白的分析。
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, alpha 2,6- and alpha 2,3-linked sialic acids are selectively amidated with different length of alkyl chains, allowing distinction of alpha 2,3-/alpha 2,6-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 alpha 2,3-/alpha 2,6-linked sialic acids compared with existing sialic acid derivatization approaches. SALSA fully stabilizes both alpha 2,3- and alpha 2,6-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.