Polysialylated N‑Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations, and Electrospray Ionization-Mass Spectrometry

The
N-glycan diversity of human serum glycoproteins, i.e., the
human blood serum N-glycome, is both complex and constrained by the
range of glycan structures potentially synthesizable by human glycosylation
enzymes. The known glycome, however, has been further limited by methods
of sample preparation, available analytical platforms, e.g., based
upon electrospray ionization-mass spectrometry (ESI-MS), and software
tools for data analysis. In this report several improvements have
been implemented in sample preparation and analysis to extend ESI-MS
glycan characterization and to include polysialylated N-glycans. Sample
preparation improvements included acidified, microwave-accelerated,
PNGase F N-glycan release to promote lactonization, and sodium borohydride
reduction, that were both optimized to improve quantitative yields
and conserve the number of glycoforms detected. Two-stage desalting
(during solid phase extraction and on the analytical column) increased
sensitivity by reducing analyte signal division between multiple reducing-end-forms
or cation adducts. Online separations were improved by using extended
length graphitized carbon columns and adding TFA as an acid modifier
to a formic acid/reversed phase gradient, providing additional resolving
power and significantly improved desorption of both large and heavily
sialylated glycans. To improve MS sensitivity and provide gentler
ionization conditions at the source-MS interface, subambient pressure
ionization with nanoelectrospray (SPIN) was utilized. When these improved
methods are combined together with the Glycomics Quintavariate Informed
Quantification (GlyQ-IQ) recently described (Kronewitter et al. Anal. Chem. 2014, 86, 6268−6276), we are able to significantly extend glycan
detection sensitivity and provide expanded glycan coverage. We demonstrated
the application of these advances in the context of the human serum
glycome, and for which our initial observations included the detection
of a new class of heavily sialylated N-glycans, including polysialylated
N-glycans.

人血清糖蛋白的N-聚糖（N-glycan）多样性，即人血清N-糖组（N-glycome），既具有复杂性，又受限于人类糖基化酶所能合成的聚糖结构范围。然而，已探明的糖组还因样品制备方法、现有分析平台（如基于电喷雾电离质谱（ESI-MS）的平台）以及数据分析软件工具的局限而未得到充分覆盖。本研究对样品制备与分析流程实施了多项改进，以拓展ESI-MS的聚糖表征能力，并实现多唾液酸化N-聚糖的检测。样品制备方面的改进包括采用酸化、微波辅助的肽N-糖苷酶F（PNGase F）法释放N-聚糖以促进内酯化，以及硼氢化钠还原步骤，上述操作均经过优化以提升定量产率并保留可检测的糖型数量。两步脱盐（分别在固相萃取阶段与分析柱上进行）通过减少多种还原端形式或阳离子加合物之间的分析物信号分流，提升了检测灵敏度。在线分离流程则通过使用长程石墨化碳柱，并在甲酸/反相梯度洗脱体系中添加三氟乙酸（TFA）作为酸性修饰剂，获得了更强的分离能力，并显著改善了大分子与高唾液酸化聚糖的解吸效果。为提升质谱灵敏度并在源-质谱接口处提供更温和的电离条件，本研究采用了纳米电喷雾亚常压电离（SPIN）技术。将上述改进方法与近期报道的糖组学五变量信息量化（GlyQ-IQ）技术（Kronewitter等人，"Anal. Chem. 2014, 86, 6268−6276"）相结合后，我们可显著提升聚糖检测灵敏度，并拓展聚糖覆盖范围。我们在人血清糖组的研究中验证了这些技术的应用效果，初步观测结果显示，我们检测到了一类新型的高唾液酸化N-聚糖，其中包括多唾液酸化N-聚糖。