Large Scale Screening and Quantitative Analysis of Site-Specific N-Glycopeptides from Human Serum in Early Alzheimer's Disease Using LC-HCD-PRM-MS.
Large Scale Screening and Quantitative Analysis of Site-Specific N-Glycopeptides from Human Serum in Early Alzheimer's Disease Using LC-HCD-PRM-MS.
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发表时间：
2022
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通讯作者：
Lubman DM
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作者：
Pan L;Lin Y;Zhu J;Zhang J;Tan Z;Lubman DM
质谱法分析糖肽可能为发现阿尔茨海默病（AD）早期检测的生物标志物提供了重要的机会。在这项工作中，我们使用nanolc - steped - hcd - dda -MS/MS平台和nanolc - steped - hcd - prm -MS平台大规模筛选和定量了用于早期检测患者血清中AD的新型n -糖肽生物标志物。通过胰蛋白酶消化、糖肽富集、分离、nanolc - steped - hcd - dda -MS/MS或nanolc - steped - hcd - prm -MS分析，分别从轻度认知障碍患者（MCI， AD前驱期）和正常人的10 μL血清中提取n -糖肽。使用Byonic， Byologic和Skyline软件的组合，我们能够在MCI和正常对照之间完成位点特异性n -糖肽的鉴定和无标记定量。Byologic的差异定量分析显示，与正常对照相比，MCI患者中来自16种糖蛋白的29种n -糖肽发生了显著变化。此外，HCD-PRM-MS对选定的n -糖肽候选物进行定量分析，证实来自CERU的EHEGAIYPDN138TTDFQR_HexNAc(4)Hex(5)-Fuc(2)NeuAc(1)和来自AHSG的VCQDCPLLAPLN156DTR_HexNAc(4)Hex(5)NeuAc(2)可以显著区分MCI和正常对照。两种糖肽的受试者工作特征曲线下面积（AUC）分别为0.850 （95% CI, 0.66 ~ 1.0）和0.867 (95% CI, 0.68 ~ 1.0) （p<0.05）。结果表明，n -糖肽表达水平的变化为早期检测AD提供了潜在的血清生物标志物。
Glycopeptide analysis by mass spectrometry may provide an important opportunity in discovery of biomarkers to aid in early detection of Alzheimer’s Disease (AD). In this work, we have used a NanoLC-Stepped-HCD-DDA-MS/MS platform and a NanoLC-Stepped-HCD-PRM-MS platform for large-scale screening and quantification of novel N-glycopeptide biomarkers for early detection of AD in patient serum. N-glycopeptides were retrieved from 10 μL of serum in patients with mild cognitive impairment (MCI, a prodromal phase of AD) and normal controls, respectively, after trypsin digestion, glycopeptide enrichment, fractionation, and NanoLC-Stepped-HCD-DDA-MS/MS or NanoLC-Stepped-HCD-PRM-MS analysis. Using a combination of Byonic, Byologic and Skyline softwares, we were able to accomplish both identification and label-free quantitation of site-specific N-glycopeptides between MCI and normal controls. Differential quantitation analysis by Byologic showed that 29 N-glycopeptides derived from 16 glycoproteins were significantly changed in MCI compared to normal controls. Further, HCD-PRM-MS quantitative analysis of the selected N-glycopeptide candidates confirmed that EHEGAIYPDN138TTDFQR_HexNAc(4)Hex(5)-Fuc(2)NeuAc(1) from CERU, and VCQDCPLLAPLN156DTR_HexNAc(4)Hex(5)NeuAc(2) from AHSG can significantly discriminate MCI from normal controls. These two glycopeptides had the area under the receiver operating characteristic curve (AUC) of 0.850 (95% CI, 0.66–1.0) and 0.867 (95% CI, 0.68–1.0), respectively (p<0.05). The result demonstrates that changes in the expression level of the N-glycopeptides provide potential serum biomarkers for detection of AD at a very early stage.