State Key Laboratory of Genetic Engineering, Department of Biochemistry and Biophysics, School of Life Sciences, Fudan University, Shanghai 200438, China.
Anal Chem. 2022 Sep 20;94(37):12565-12569. doi: 10.1021/acs.analchem.2c02099. Epub 2022 Sep 6.
Isobaric labeling is the most widely used multiplexing quantitative approach in proteomic studies, enabling the comparison of up to 18 samples in a single MS analysis. Expanding the multiplexing capacity is of great necessity for high-throughput proteomic studies. Herein, we establish a novel TAG-TMTpro approach by introducing Ala or Gly residues to peptides prior to TMTpro labeling, which is able to triple the quantitative capacity of TMTpro. We systematically evaluated the Boc-Ala-OSu and Boc-Gly-OSu reaction and optimized the conditions for labeling, side-product elimination, and Boc deprotection. We validated the identification and quantification performance using and HeLa cell lysates. We demonstrated that the TAG-TMTpro approach resulted in good identification reproducibility and reliable quantitative accuracy. The TAG-TMTpro is able to triple the multiplexing capacity of TMTpro reagents and is a versatile quantitative approach for high-throughput proteomic studies. Data are available via ProteomeXchange with identifier PXD033711.
同位素标记是蛋白质组学研究中最广泛使用的多重定量方法，可在单次 MS 分析中比较多达 18 个样本。为了进行高通量蛋白质组学研究，扩展多重化容量是非常必要的。在此，我们通过在 TMTpro 标记之前在肽中引入 Ala 或 Gly 残基，建立了一种新的 TAG-TMTpro 方法，从而将 TMTpro 的定量能力提高了三倍。我们系统地评估了 Boc-Ala-OSu 和 Boc-Gly-OSu 反应，并优化了标记、副产物消除和 Boc 脱保护的条件。我们使用 和 HeLa 细胞裂解物验证了鉴定和定量性能。我们证明，TAG-TMTpro 方法具有良好的鉴定重现性和可靠的定量准确性。TAG-TMTpro 能够将 TMTpro 试剂的多重化容量提高三倍，是一种用于高通量蛋白质组学研究的通用定量方法。数据可通过 ProteomeXchange 以标识符 PXD033711 获得。
