Division of Pharmaceutical Analysis, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 645 South Newstead Avenue, St. Louis, Missouri 63110, United States.
Anal Chem. 2020 Jun 2;92(11):7547-7555. doi: 10.1021/acs.analchem.0c00014. Epub 2020 May 14.
A recently developed synchronous precursor selection (SPS) mass spectrometry to the third (MS3) protocol enables more accurate multiplexed quantification of proteins/peptides using tandem mass tags (TMT) through comparison of reporter ion intensities at the MS3 level. However, challenges still exist for TMT-based simultaneous quantification and identification of intact glycopeptides due to inefficient peptide backbone fragmentation when using collision-induced dissociation (CID). To overcome this limitation, here we report an improved SPS/ETD workflow for TMT-based intact glycopeptide quantification and identification. The SPS/ETD approach was implemented on an Orbitrap Tribrid mass spectrometer and begins with selection of a parent ion in the MS scan, followed by tandem mass spectrometry (MS2) fragmentation by CID in the ion trap. Following MS2 fragmentation, SPS enables simultaneous isolation of the top 10 MS2 fragment ions for further higher energy collisional dissociation (HCD) fragmentation with the resulting MS3 fragments detected in an Orbitrap analyzer. Here, in addition to the standard SPS workflow, an electron-transfer dissociation (ETD) MS2 was performed and analyzed in the ion trap. The resultant ETD and CID spectra were used for the identification of the intact glycopeptides, while the quantitative comparison of site-specific glycans was achieved utilizing TMT reporter ions from HCD MS3 spectra. For intact glycopeptides, through systematic optimization and evaluation using a glycoprotein interference model, the SPS/ETD approach was demonstrated to offer improved accuracy, precision, and sensitivity compared to traditional data-dependent MS2 quantification, while maintaining the glycopeptide identification capability. Finally, this workflow was applied for the site-specific quantitative comparison of the glycoforms for two therapeutic enzymes (Cerezyme and VPRIV) and their different lots. The results demonstrate that this workflow is suitable for TMT-based intact glycopeptide characterization of glycoproteins.
最近开发的同步前体选择（SPS）质谱法到第三级（MS3）协议，通过在 MS3 水平比较报告离子强度，能够更准确地对串联质量标签（TMT）进行蛋白质/肽的多重定量。然而，由于在使用碰撞诱导解离（CID）时肽主链的碎片化效率低下，基于 TMT 的完整糖肽的同时定量和鉴定仍然存在挑战。为了克服这一限制，我们在此报告了一种用于基于 TMT 的完整糖肽定量和鉴定的改进的 SPS/ETD 工作流程。SPS/ETD 方法在轨道阱三重四极杆质谱仪上实施，首先在 MS 扫描中选择母离子，然后在离子阱中通过 CID 进行串联质谱（MS2）碎片化。在 MS2 碎片化之后，SPS 能够同时分离前 10 个 MS2 碎片离子，以便进一步用高能碰撞解离（HCD）进行碎片化，在轨道阱分析仪中检测到 MS3 碎片。在这里，除了标准的 SPS 工作流程外，还在离子阱中进行了电子转移解离（ETD）MS2 并进行了分析。使用 ETD 和 CID 谱进行完整糖肽的鉴定，而利用 HCD MS3 谱的 TMT 报告离子实现了特定位点糖基的定量比较。对于完整糖肽，通过使用糖蛋白干扰模型进行系统优化和评估，与传统的基于数据依赖的 MS2 定量相比，SPS/ETD 方法显示出更高的准确性、精度和灵敏度，同时保持糖肽鉴定能力。最后，该工作流程用于两种治疗酶（Cerezyme 和 VPRIV）及其不同批次的糖型的特定位点定量比较。结果表明，该工作流程适用于基于 TMT 的糖蛋白完整糖肽特征。
