Benefiting from high sensitivity and great ability to measure multiple samples simultaneously, isobaric tandem Mass spectrometry (MS2) quantification has been widely applied for protein biomarker screening. Here, a newly developed isobaric MS2 quantification method named triplex quantification by isobaric termini labeling (Triplex-QITL) was established. This method enables the accurate comparison of various fragment ions (reporter ions, amino acid fragments and N-/C-terminal fragments) based quantification to be operated in a single run. To our knowledge, this is the first time that this kind of comparison is achieved. In Triplex-QITL, proteins were first digested with Lys-C to produce peptides with lysine (K) at the C-termini, then dimethylation reagents and mTRAQ reagents were used to label the N-termini and C-termini of the peptides respectively. N- and C-terminal fragment ion pairs, reporter ions from mTRAQ (113,117,121) and a1 ion pairs were simultaneously generated in MS2 spectra. In simple sample experiment, not much difference in using various fragment ions for quantification was observed. When analyzing SW480 cell lysate, comparing with a1 ions, about two times of reproducible quantification results were achieved by reporter ions and N- and C-terminal ions. Meanwhile the measured quantification results were much closer to the expected results even in large ratios (1:10:10) using N- and C-terminal ions. Finally, Triplex-QITL was successfully applied to profile metastatic differences of three hepatocellular carcinoma (HCC) cell lines. In all, Triplex-QITL shows a promising future in quantitative proteomics.

中文翻译：

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一种新的三重等压末端标记定量方法，可同时提供三种定量来源

受益于高灵敏度和同时测量多个样品的强大能力，等压串联质谱 (MS2) 定量已广泛应用于蛋白质生物标志物筛选。在这里，建立了一种新开发的等压 MS2 定量方法，称为等压末端标记三重定量 (Triplex-QITL)。这种方法能够在一次运行中准确比较各种碎片离子（报告离子、氨基酸碎片和 N-/C-末端碎片）。据我们所知，这是第一次实现这种比较。在 Triplex-QITL 中，蛋白质首先用 Lys-C 消化以产生在 C 端带有赖氨酸 (K) 的肽，然后用二甲基化试剂和mTRAQ试剂分别标记肽的N-末端和C-末端。N 端和 C 端碎片离子对、来自 mTRAQ (113,117,121) 的报告离子和 a1 离子对在 MS2 光谱中同时生成。在简单的样品实验中，观察到使用各种碎片离子进行定量并没有太大差异。在分析 SW480 细胞裂解液时，与 a1 离子相比，报告离子和 N 端和 C 端离子实现了约两倍可重现的定量结果。同时，即使在使用 N 端和 C 端离子的大比例 (1:10:10) 下，测量的定量结果也更接近预期结果。最后，Triplex-QITL 成功应用于分析三种肝细胞癌 (HCC) 细胞系的转移差异。在所有，