Wang Benlian, Tsybovsky Yaroslav, Palczewski Krzysztof, Chance Mark R
Center for Proteomics and Bioinformatics, School of Medicine, Case Western Reserve University, Cleveland, OH, 44106, USA.
J Am Soc Mass Spectrom. 2014 May;25(5):729-41. doi: 10.1007/s13361-013-0823-6. Epub 2014 Feb 19.
Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.
通过质谱同时对聚糖和肽段进行表征的位点特异性糖肽图谱分析具有难度，这是因为蛋白质糖基化具有异质性和多样性，并且利用当前的碎裂技术，肽段或聚糖都缺乏完整的碎裂信息。实际上，即使质量误差小于5 ppm，多种肽段和聚糖组合也很容易与相同质量的糖肽匹配，这会带来相当大的歧义性，对于大蛋白而言，分析复杂的糖肽混合物极具挑战性。在此，我们报告了一种新策略，通过将仅碰撞诱导解离（CID）碎裂与糖肽的色谱保留时间相结合，可靠地确定位点特异性N-糖基化图谱。该方法利用了一种对糖肽和去糖肽进行平行分析的实验流程。作为测试案例，我们选择了ABCA4，这是一种大型整合膜蛋白，预测有16个N-糖基化位点。利用CID的特征，如高扫描速度和高碎片离子强度，再结合糖肽的保留时间，以最终确定糖肽所源自的非糖基化肽段，我们获得了关于聚糖组成、肽段序列以及ABCA4特定位点的N-糖基化位点占有率和每种糖型相对丰度的几乎完整的信息。该案例所带来的挑战为分析复杂的相对纯的糖蛋白以及潜在的更复杂的糖蛋白混合物提供了指导。
