Isotope-Coded Carbamidomethylation for Quantification of N-Glycoproteins with Online Microbore Hollow Fiber Enzyme Reactor-Nanoflow Liquid Chromatography-Tandem Mass Spectrometry
Isotope-Coded Carbamidomethylation for Quantification of N-Glycoproteins with Online Microbore Hollow Fiber Enzyme Reactor-Nanoflow Liquid Chromatography-Tandem Mass Spectrometry
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DOI：
10.1021/ac501544r
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发表时间：
2014-08-05
影响因子：
7.4
通讯作者：
Moon, Myeong Hee
中科院分区：
文献类型：
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作者：
Kim, Jin Yong;Oh, Donggeun;Moon, Myeong Hee
本文介绍了一种简单、廉价和可靠的N-连接糖蛋白定量蛋白质组学方法，该方法基于在线微孔中空纤维酶反应器中的同位素编码的氨基甲基化(ICCM)和纳米流液相色谱-串联质谱仪(mHFER-NLC-MS/MS)。ICCM定量使用蛋白质的半胱氨酸残基与碘乙酰胺(IAA)或其同位素(IAA-C-13(2)，D-2：4 Da差)的氨基甲基化(CM；蛋白分解前硫醇基团的常规保护)。CM-/ICCM标记的蛋白质组样品混合用于蛋白质降解；然后，利用凝集素亲和力在mHFER中进行在线浓缩N-糖肽，然后使用NLC-MS/MS进行多反应监测(MRM)定量。改变CM-/ICCM标记的牛血清白蛋白(BSA)标准品的混合比例的ICCM方法的初步评估产生了18个多肽的成功定量，计算的轻/重标记多肽的比例变化小于2%。用三种标准糖蛋白(α-1-酸性糖蛋白、胎球蛋白和转铁蛋白)对mHFER-NLC-MS/MS进行ICCM定量，并应用于肝癌患者和对照组的血清糖蛋白，成功地定量了49个N-糖蛋白中的73个N-糖肽，其中来自14个N-糖蛋白的19个N-糖肽的异常变化是对照的2.5倍以上。虽然mHFER-NLC-MS/MS的ICCM定量只适用于含有半胱氨酸残基的糖肽，但与其他标记方法相比，该方法在用于靶向糖蛋白时具有几个优点：ICCM方法不需要在特殊条件下进行额外的标记反应，也不需要使用额外的柱来纯化标记产品的复杂程序。在蛋白质水平上进行同位素标记可以最大限度地减少因在单独的小瓶中消化蛋白质和在多肽水平上进行标记时检索每个标记的多肽的效率不同而产生的潜在不确定性。此外，ICCM方法的标记试剂很容易以合理的价格获得，这使得蛋白质定量变得容易。
This paper introduces a simple, inexpensive, and robust quantitative proteomic method for quantifying N-linked glycoproteins based on isotope-coded carbamidomethylation (iCCM) incorporated into an online microbore hollow fiber enzyme reactor and nanoflow liquid chromatography-tandem mass spectrometry (mHFER-nLC-MS/MS). The iCCM quantitation uses carbamidomethylation (CM; a routine protection of thiol groups before proteolysis) of the Cys residue of proteins with iodoacetamide (IAA) or its isotope (IAA-C-13(2),D-2: 4 Da difference). CM-/iCCM-labeled proteome samples are mixed for proteolysis; then, online enrichment of N-glycopeptides using lectin affinity is carried out in an mHFER before nLC-MS/MS for quantification using multiple reaction monitoring (MRM). Initial evaluation of the iCCM method varying the mixing ratio of CM-/iCCM-labeled bovine serum albumin (BSA) standards yielded successful quantification of 18 peptides with less than 2% variation in the calculated ratio of light/heavy-labeled peptides. The iCCM quantitation with mHFER-nLC-MS/MS was evaluated with three standard glycoproteins (alpha-1-acid glycoproteins, fetuin and transferrin) and then applied to serum glycoproteins from liver cancer patients and controls, resulting in successful quantification of 73 N-glycopeptides (from 49 N-glycoproteins), among which 19 N-glycopeptides from 14 N-glycoproteins showed more than a 2.5-fold aberrant change in liver cancer patients' sera compared with the pooled control. Although iCCM quantitation with mHFER-nLC-MS/MS applies only to glycopeptides with Cys residue, the method can offer several advantages over other labeling methods when applied to targeted glycoproteins: The iCCM method does not require an additional labeling reaction under special conditions nor complicated procedures to purify labeled products using additional columns. Isotope labeling at the protein level can minimize potential uncertainty originating from unequal efficiencies in protein digestion in separate vials and retrieval of each labeled peptide when labeling takes place at the peptide level. In addition, the labeling reagents for the iCCM method are readily obtained at a reasonable cost, which can make protein quantification easily accessible.