Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.
Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.
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基于全甲基化硫酸聚糖的质谱图谱和测序的磺基糖组学的启用技术和战略工作流程。
作者：
Shin‐Yi Yu;Sz;H. Hsiao;K. Khoo
N-和O-聚糖末端表位上的硫酸盐修饰越来越被认为是介导多种生物识别功能的关键决定因素。为了解决这些低丰度但重要的硫酸化聚糖和一般的磺基糖组，需要进一步开发富集策略和基于质谱 (MS) 的作图技术。在本报告中，我们证明，无论有或没有额外的唾液酸化，硫酸化聚糖都可以通过氢氧化钠浆液法成功地全甲基化，并通过这种衍生化与磷酸化聚糖区分开来。结合简单的微量衍生化后分馏步骤，完全保留带负电荷的硫酸盐部分并与非硫酸盐部分分离的全甲基衍生物，可以通过先进的 MS/MS 在正离子模式下进行有效的检测和从头测序。特别是，我们表明，MALDI-TOF/TOF 提供的高度序列和连锁信息的高能碰撞诱导解离 (CID) MS/MS 可以扩展到磺糖组学应用。发现为 CID MS/MS 选择的硫酸化母离子大部分保留了硫酸盐部分，因此可以通过通常的糖苷、交叉环和协同双裂解阵列进行有效裂解。总的来说，优化的策略能够对磺基糖组进行高灵敏度检测和关键图谱，例如在负离子和正离子模式下源自淋巴结组织或细胞系的磺基糖组。从粗制小鼠淋巴结制剂中鉴定出新的硫酸化表位，这充分证明了所开发方法的实用性。
Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed.
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影响因子：
7.4
作者：
Lapadula, AJ;Hatcher, PJ;Reinhold, VN
通讯作者：
Reinhold, VN
影响因子：
7.4
作者：
Ashline, D;Singh, S;Reinhold, V
通讯作者：
Reinhold, V
影响因子：
7.4
作者：
Ashline, David J.;Lapadula, Anthony J.;Reinhold, Vernon N.
通讯作者：
Reinhold, Vernon N.
影响因子：
2
作者：
Kang, Pilsoo;Mechref, Yehia;Novotny, Milos V.
通讯作者：
Novotny, Milos V.
影响因子：
--
作者：
Zaia, Joseph
通讯作者：
Zaia, Joseph