Yang Hong, Yang Chenxi, Sun Taolei
State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, 122 Luoshi Road, Wuhan, 430070, China.
School of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, 122 Luoshi Road, Wuhan, 430070, China.
Rapid Commun Mass Spectrom. 2018 Aug 30;32(16):1353-1362. doi: 10.1002/rcm.8191.
Accurate characterization of glycopeptides without a prior glycan cleavage could provide valuable information on site-specific glycosylation, which is critical to reveal the biological functions of protein glycosylation. However, due to the distinct nature of oligosaccharides and ploypeptides, it is usually difficult to effectively fragment glycopeptides in mass spectrometry analysis.
Here we applied a stepped normalized collisional energy (NCE) approach, which is able to combine fragment ions from three different collision energies, in a hybrid quadrupole orbitrap (Q Exactive Plus) to characterize glycopeptides. A systematic evaluation was firstly performed to find optimal NCE values for the fragmentation of glycan chains and peptide backbones from glycopeptides. Guided by the results of the systematic evaluation, the stepped NCE method was optimized and employed to analyze glycopeptides enriched from human serum.
The stepped NCE approach was found to effectively fragment both the glycan chains and peptide backbones from glycopeptides and record these fragments in a single MS/MS spectrum. In comparison with the regular HCD methods, the stepped NCE method identified more glycopeptides with higher scores from human serum samples.
Our studies demonstrate the capability of stepped NCE for the effective characterization of glycopeptides on a large scale.
在不预先进行聚糖切割的情况下对糖肽进行准确表征，可以提供关于位点特异性糖基化的有价值信息，这对于揭示蛋白质糖基化的生物学功能至关重要。然而，由于寡糖和多肽的性质不同，在质谱分析中通常难以有效地裂解糖肽。
在此，我们应用了一种阶梯式归一化碰撞能量（NCE）方法，该方法能够在混合四极杆轨道阱（Q Exactive Plus）中结合来自三种不同碰撞能量的碎片离子，以表征糖肽。首先进行了系统评估，以找到用于裂解糖肽聚糖链和肽主链的最佳NCE值。在系统评估结果的指导下，对阶梯式NCE方法进行了优化，并用于分析从人血清中富集的糖肽。
发现阶梯式NCE方法能够有效地裂解糖肽的聚糖链和肽主链，并在单个MS/MS谱图中记录这些碎片。与常规的高能碰撞解离（HCD）方法相比，阶梯式NCE方法从人血清样品中鉴定出更多得分更高的糖肽。
我们的研究证明了阶梯式NCE大规模有效表征糖肽的能力。
