Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
Talanta. 2018 Mar 1;179:22-27. doi: 10.1016/j.talanta.2017.10.015. Epub 2017 Oct 31.
Glycoproteomic analysis requires efficient separation and sensitive detection to enable the comprehensive characterization of glycan heterogeneity. Here, we report the use of capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) with an electrokinetically-pumped nanospray interface for the study of protein glycosylation microheterogeneity. A fast separation was developed that resolved intact glycopeptides generated from standard proteins within ~9min. Differentially terminal-galactosylated and sialylated species with the same glycosylation sites were well resolved. The concentration detection limits for CZE were three times higher than for nanoLC methods; however, a 200-fold smaller injection volume was used in CZE, which reflects the use of an extremely efficient electrospray interface in our CZE-ESI-MS setup. The resulting glycopeptide mass detection limit was two orders of magnitude superior to a nanoLC method. We also observed a 1.5% and 7% average relative standard deviation in peak migration time and glycopeptide relative abundance, and a four order of magnitude linear dynamic range in signal intensity. With CZE-ESI-MS, 40 haptoglobin glycopeptides were identified from roughly 40 fmol of digest.
糖蛋白质组学分析需要有效的分离和灵敏的检测，以实现对糖基异质性的全面描述。在这里，我们报告了使用毛细管区带电泳-电喷雾电离-质谱（CZE-ESI-MS）与电动泵纳米喷雾接口来研究蛋白质糖基化微异质性。开发了一种快速分离方法，可在约 9 分钟内分离出标准蛋白质产生的完整糖肽。具有相同糖基化位点的不同末端半乳糖基化和唾液酸化的物质得到了很好的分离。CZE 的浓度检测限比纳诺 LC 方法高 3 倍；然而，CZE 中使用的进样体积小 200 倍，这反映了我们在 CZE-ESI-MS 装置中使用了极其高效的电喷雾接口。所得糖肽质量检测限比纳诺 LC 方法高两个数量级。我们还观察到峰迁移时间和糖肽相对丰度的平均相对标准偏差分别为 1.5%和 7%，信号强度的线性动态范围为四个数量级。通过 CZE-ESI-MS，从大约 40 飞摩尔的消化物中鉴定出了 40 种触珠蛋白糖肽。
