Yu Shin-Yi, Snovida Sergei, Khoo Kay-Hooi
Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan.
University Lille, CNRS, UMR 8576 -UGSF- Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France.
Bio Protoc. 2020 May 20;10(10):e3617. doi: 10.21769/BioProtoc.3617.
Sulfated glycans are barely detectable in routine mass spectrometry (MS)-based glycomic analysis due to ion suppression by the significantly more abundant neutral glycans in the positive ion mode, and sialylated non-sulfated glycans in the negative ion mode, respectively. Nevertheless, the negative charge imparted by sulfate can be advantageous for selective detection in the negative ion mode if the sialic acids can first be neutralized. This is most conveniently achieved by a concerted sample preparation workflow in which permethylation is followed by solid phase fractionation to isolate the sulfated glycans prior to MS analysis. Importantly, we demonstrated that conventional NaOH/DMSO slurry permethylation method can retain the sulfates. Instead of extracting permethylated glycans into chloroform for sample clean-up, reverse phase C18 cartridge coupled with self-packed amine-tip or mixed mode weak anion exchange cartridge can be utilized to obtain in good yield the non-sulfated, mono-sulfated, and multiply sulfated permethylated glycans in separate fractions for sulfoglycomic analysis.
在基于常规质谱（MS）的糖组学分析中，硫酸化聚糖很难被检测到，这是因为在正离子模式下，大量存在的中性聚糖会产生离子抑制作用；而在负离子模式下，唾液酸化的非硫酸化聚糖会产生离子抑制作用。然而，如果唾液酸首先被中和，硫酸基团所赋予的负电荷对于在负离子模式下的选择性检测可能是有利的。这可以通过一个协同的样品制备流程最方便地实现，其中在进行质谱分析之前，先进行全甲基化，然后通过固相分级分离来分离硫酸化聚糖。重要的是，我们证明了传统的NaOH/DMSO浆液全甲基化方法可以保留硫酸盐。无需将全甲基化的聚糖萃取到氯仿中进行样品净化，而是可以使用反相C18柱结合自装胺尖或混合模式弱阴离子交换柱，以良好的产率分别获得非硫酸化、单硫酸化和多硫酸化的全甲基化聚糖馏分，用于硫酸化糖组学分析。
