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Selective reaction monitoring approach using structure-defined synthetic glycopeptides for validating glycopeptide biomarkers pre-determined by bottom-up glycoproteomics
RSC Advances ( IF 3.9 ) Pub Date : 2022-08-03 , DOI: 10.1039/d2ra02903k Kouta Shiratori 1 , Yasuhiro Yokoi 2 , Hajime Wakui 1 , Nozomi Hirane 1 , Michiru Otaki 1 , Hiroshi Hinou 1 , Tohru Yoneyama 3 , Shingo Hatakeyama 3 , Satoshi Kimura 4 , Chikara Ohyama 3 , Shin-Ichiro Nishimura 1, 2
RSC Advances ( IF 3.9 ) Pub Date : 2022-08-03 , DOI: 10.1039/d2ra02903k Kouta Shiratori 1 , Yasuhiro Yokoi 2 , Hajime Wakui 1 , Nozomi Hirane 1 , Michiru Otaki 1 , Hiroshi Hinou 1 , Tohru Yoneyama 3 , Shingo Hatakeyama 3 , Satoshi Kimura 4 , Chikara Ohyama 3 , Shin-Ichiro Nishimura 1, 2
Affiliation
Clusterin is a heavily glycosylated protein that is upregulated in various cancer and neurological diseases. The findings by the Hancock and Iliopoulos group that levels of the tryptic glycopeptide derived from plasma clusterin, 372Leu-Ala-Asn-Leu-Thr-Gln-Gly-Glu-Asp-Gln-Tyr-Tyr-Leu-Arg385 with a biantennary disialyl N-glycan (A2G2S2 or FA2G2S2) at Asn374 differed significantly prior to and after curative nephrectomy for clear cell renal cell carcinoma (RCC) patients motivated us to verify the feasibility of this glycopeptide as a novel biomarker of RCC. To determine the precise N-glycan structure attached to Asn374, whether A2G2S2 is composed of the Neu5Acα2,3Gal or/and the Neu5Acα2,6Gal moiety, we synthesized key glycopeptides having one of the two putative isomers. Selective reaction monitoring assay using synthetic glycopeptides as calibration standards allowed “top-down glycopeptidomics” for the absolute quantitation of targeted label-free glycopeptides in a range from 313.3 to 697.5 nM in the complex tryptic digests derived from serum samples of RCC patients and healthy controls. Our results provided evidence that the Asn374 residue of human clusterin is modified dominantly with the Neu5Acα2,6Gal structure and the levels of clusterin bearing an A2G2S2 with homo Neu5Acα2,6Gal terminals at Asn374 decrease significantly in RCC patients as compared with healthy controls. The present study elicits that a new strategy integrating the bottom-up glycoproteomics with top-down glycopeptidomics using structure-defined synthetic glycopeptides enables the confident identification and quantitation of the glycopeptide targets pre-determined by the existing methods for intact glycopeptide profiling.
中文翻译:
使用结构定义的合成糖肽的选择性反应监测方法来验证由自下而上的糖蛋白质组学预先确定的糖肽生物标志物
簇蛋白是一种高度糖基化的蛋白质,在各种癌症和神经系统疾病中表达上调。第372章_ _在透明细胞肾细胞癌 (RCC) 患者的根治性肾切除术之前和之后,Asn374 处的双触角二唾液酰 N-聚糖(A2G2S2 或 FA2G2S2)存在显着差异,这促使我们验证这种糖肽作为 RCC 新型生物标志物的可行性。为了确定连接到 Asn374 的精确N -聚糖结构,A2G2S2 是否由 Neu5Acα2,3Gal 或/和 Neu5Acα2,6Gal 部分组成,我们合成了具有两种假定异构体之一的关键糖肽。使用合成糖肽作为校准标准的选择性反应监测分析允许“自上而下的糖肽组学”对来自 RCC 患者和健康对照血清样本的复杂胰蛋白酶消化物中 313.3 至 697.5 nM 范围内的目标无标记糖肽进行绝对定量。我们的结果提供了证据,表明人类凝聚素的 Asn374 残基主要被 Neu5Acα2,6Gal 结构修饰,并且与健康对照相比,RCC 患者中 Asn374 处带有同型 Neu5Acα2,6Gal 末端的 A2G2S2 的凝聚素水平显着降低。本研究表明,使用结构定义的合成糖肽将自下而上的糖蛋白质组学与自上而下的糖肽组学相结合的新策略能够可靠地识别和定量通过现有的完整糖肽分析方法预先确定的糖肽靶点。
更新日期:2022-08-03
中文翻译:
使用结构定义的合成糖肽的选择性反应监测方法来验证由自下而上的糖蛋白质组学预先确定的糖肽生物标志物
簇蛋白是一种高度糖基化的蛋白质,在各种癌症和神经系统疾病中表达上调。第372章_ _在透明细胞肾细胞癌 (RCC) 患者的根治性肾切除术之前和之后,Asn374 处的双触角二唾液酰 N-聚糖(A2G2S2 或 FA2G2S2)存在显着差异,这促使我们验证这种糖肽作为 RCC 新型生物标志物的可行性。为了确定连接到 Asn374 的精确N -聚糖结构,A2G2S2 是否由 Neu5Acα2,3Gal 或/和 Neu5Acα2,6Gal 部分组成,我们合成了具有两种假定异构体之一的关键糖肽。使用合成糖肽作为校准标准的选择性反应监测分析允许“自上而下的糖肽组学”对来自 RCC 患者和健康对照血清样本的复杂胰蛋白酶消化物中 313.3 至 697.5 nM 范围内的目标无标记糖肽进行绝对定量。我们的结果提供了证据,表明人类凝聚素的 Asn374 残基主要被 Neu5Acα2,6Gal 结构修饰,并且与健康对照相比,RCC 患者中 Asn374 处带有同型 Neu5Acα2,6Gal 末端的 A2G2S2 的凝聚素水平显着降低。本研究表明,使用结构定义的合成糖肽将自下而上的糖蛋白质组学与自上而下的糖肽组学相结合的新策略能够可靠地识别和定量通过现有的完整糖肽分析方法预先确定的糖肽靶点。