Multidimensional separation and analysis of alpha-1-acid glycoprotein N-glycopeptides using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and nano-liquid chromatography tandem mass spectrometry.
Multidimensional separation and analysis of alpha-1-acid glycoprotein N-glycopeptides using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and nano-liquid chromatography tandem mass spectrometry.
复制标题
使用高场不对称波形离子迁移谱 (FAIMS) 和纳米液相色谱串联质谱法对 α-1-酸性糖蛋白 N-糖肽进行多维分离和分析。
DOI：
10.1007/s00216-022-04435-3
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发表时间：
2023
影响因子：
4.3
通讯作者：
Sackstein,Robert
中科院分区：
文献类型：
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作者：
Chandler,KevinBrown;MarreroRoche,DanielE;Sackstein,Robert
自下而上的基于nLC-MS/MS的糖蛋白质谱工作流程依赖于通过糖蛋白的蛋白水解生成非糖基化和糖基化肽的混合物。这些方法受到更丰富的、疏水的和容易电离的非糖基化肽对亲水性糖肽离子的抑制的挑战。最近已经引入了市售的高场不对称波形离子迁移谱（FAIMS）装置，并且通过在色谱分离之后并且在MS/MS分析之前实现非糖基化肽和糖肽的正交分离，为糖蛋白组学工作流程提供了潜在益处。然而，知识是缺乏最佳FAIMS条件的糖肽分析。在这里，我们记录最佳FAIMS补偿电压的传输和分析的人α-1-酸性糖蛋白（AGP）胰蛋白酶N-糖肽离子。此外，我们通过使用标准nLC-MS/MS方法或采用FAIMS的其他相同方法在不同置信水平下比较分配的糖肽数量，评估FAIMS对AGP糖肽分配置信度的影响。优化的方法将通过增加独特糖肽鉴定的数量和糖肽分配的置信度来加强糖蛋白组学分析。数据可通过ProteomeXchange获得，标识符为PXD 036667。图形摘要通过nLC-FAIMS-MS/MS（上）分析α-1-酸性糖蛋白（AGP）胰蛋白酶解酶，建立了分析AGPN-糖肽（下）的理想FAIMS电压，表明FAIMS可以提高糖蛋白组表征的深度。CV幅值对沿x轴沿着显示
Bottom-up nLC-MS/MS-based glycoprotein mass spectrometry workflows rely on the generation of a mixture of non-glycosylated and glycosylated peptides via proteolysis of glycoproteins. Such methods are challenged by suppression of hydrophilic glycopeptide ions by more abundant, hydrophobic, and readily ionizable non-glycosylated peptides. Commercially available high-field asymmetric waveform ion mobility spectrometry (FAIMS) devices have recently been introduced and present a potential benefit for glycoproteomic workflows by enabling orthogonal separation of non-glycosylated peptides and glycopeptides following chromatographic separation, and prior to MS/MS analysis. However, knowledge is lacking regarding optimal FAIMS conditions for glycopeptide analyses. Here, we document optimal FAIMS compensation voltages for the transmission and analysis of human alpha-1-acid glycoprotein (AGP) trypticN-glycopeptide ions. Further, we evaluate the effect of FAIMS on AGP glycopeptide assignment confidence by comparing the number of assigned glycopeptides at different confidence levels using a standard nLC-MS/MS method or an otherwise identical method employing FAIMS. Optimized methods will potentiate glycoproteomic analyses by increasing the number of unique glycopeptide identifications and the confidence of glycopeptide assignments. Data are available via ProteomeXchange with identifier PXD036667.Graphical AbstractAnalysis of alpha-1-acid glycoprotein (AGP) tryptic digests via nLC-FAIMS-MS/MS (top) led to the establishment of ideal FAIMS voltages for the analysis of AGPN-glycopeptides (bottom), suggesting that FAIMS can improve the depth of glycoproteome characterization. Pairs of CV magnitudes are shown along thex-axis