Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, D.C. 20057, United States.
Clinical and Translational Glycoscience Research Center, Georgetown University, Washington, D.C. 20057, United States.
J Proteome Res. 2023 Apr 7;22(4):1138-1147. doi: 10.1021/acs.jproteome.2c00475. Epub 2023 Feb 10.
Targeted quantification of proteins is a standard methodology with broad utility, but targeted quantification of glycoproteins has not reached its full potential. The lack of optimized workflows and isotopically labeled standards limits the acceptance of glycoproteomics quantification. In this work, we introduce an efficient and streamlined chemoenzymatic synthesis of a library of isotopically labeled glycopeptides of IgG1 which we use for quantification in an energy optimized LC-MS/MS-PRM workflow. Incorporation of the stable isotope labeled N-acetylglucosamine enables an efficient monitoring of all major fragment ions of the glycopeptides generated under the soft higher-energy C-trap dissociation (HCD) conditions, which reduces the coefficients of variability (CVs) of the quantification to 0.7-2.8%. Our results document, for the first time, that the workflow using a combination of stable isotope labeled standards with intrascan normalization enables quantification of the glycopeptides by an electron transfer dissociation (ETD) workflow, as well as the HCD workflow, with the highest sensitivity compared to traditional workflows. This was exemplified by a rapid quantification (13 min) of IgG1 Fc glycoforms from COVID-19 patients.
靶向定量蛋白质是一种具有广泛用途的标准方法，但糖蛋白的靶向定量尚未充分发挥其潜力。缺乏优化的工作流程和同位素标记标准限制了糖蛋白质组学定量的接受程度。在这项工作中，我们介绍了一种高效、简化的化学酶合成 IgG1 糖肽同位素标记文库的方法，我们将其用于能量优化的 LC-MS/MS-PRM 工作流程中的定量。稳定同位素标记的 N-乙酰葡萄糖胺的掺入可有效地监测在软质更高能量 C 阱解离 (HCD) 条件下生成的糖肽的所有主要碎片离子，从而将定量的变异系数 (CV) 降低到 0.7-2.8%。我们的结果首次证明，使用稳定同位素标记标准品与内扫描归一化相结合的工作流程，可通过电子转移解离 (ETD) 工作流程以及 HCD 工作流程对糖肽进行定量，与传统工作流程相比，其灵敏度最高。这通过从 COVID-19 患者中快速定量 (13 分钟) IgG1 Fc 糖型得到了例证。
