Site-Specific Quantification of Protein Palmitoylation by Cysteine-Stable Isotope Metabolic Labeling.
Site-Specific Quantification of Protein Palmitoylation by Cysteine-Stable Isotope Metabolic Labeling.
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通过半胱氨酸稳定同位素代谢标记对蛋白质棕榈酰化进行位点特异性定量。
DOI：
10.1021/acs.analchem.8b02635
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发表时间：
2018
影响因子：
7.4
通讯作者：
Lu Haojie
中科院分区：
文献类型：
--
作者：
Zhang Xiaoqin;Zhang Lei;Ji Guanghui;Lei Qunying;Fang Caiyun;Lu Haojie
棕榈酰化是蛋白质翻译修饰中最重要的修饰之一，在许多重要的生物学过程中起着重要的作用。异常棕榈酰化与多种人类疾病有关。因此，对棕榈酰化蛋白质组进行定性和定量分析具有重要意义。在这里，我们描述了一种新的方法的基础上的半胱氨酸稳定同位素标记的细胞培养（半胱氨酸-SILAC），以促进定量的棕榈酰化蛋白质的质谱（MS），其中“轻”或“重”的样品可以合并，并进行随后的分析程序，同时，最大限度地减少系统误差所造成的平行操作，提高定量的准确性和精密度。质量标签位于半胱氨酸残基上，半胱氨酸残基是潜在的棕榈酰化位点，这表明可以定量所有推定的修饰位点/肽，包括一种蛋白质的C-末端肽。由于半胱氨酸的同位素标记，非特异性吸附的肽段在质谱图中为单峰，而成对峰则为棕榈酰化肽段的信号，从而降低了谱图的复杂性，实现了对棕榈酰化肽段的双重验证。最后，首次分析了具有不同转移潜能的肝细胞癌（HCC）细胞（MHCC-97 L和HCC-LM 3细胞）中的棕榈酰组。共发现151个蛋白质发生了高置信度的差异棕榈酰化，其中包括许多重要的蛋白质，这些蛋白质参与了多种生物学过程，如蛋白质棕榈酰化、细胞增殖、信号转导、细胞迁移调控等。
Palmitoylation is one of the most important protein translational modifications and plays vital roles in many key biological processes. Aberrant palmitoylation has been associated with a variety of human diseases. So it is of great significance to profile the palmitoylated proteomes qualitatively and quantitatively. Here, we described a novel method based on the cysteine-stable isotope labeling in cell culture (cysteine-SILAC) to facilitate the quantitation of palmitoylated proteins by mass spectrometry (MS), in which "light" or "heavy" samples could be pooled and subjected to the subsequent analysis procedures simultaneously, minimizing systematic errors caused by parallel operations and improving quantitative accuracy and precision. The mass tags lay on the cysteine residues, which were the potential palmitoylated sites, indicating that all the putative modified sites/peptides could be quantified, including the C-terminal peptide of one protein. Due to the isotopically labeled cysteine, the nonspecifically adsorbed peptide without cysteine was singlet in MS spectra, whereas pair peaks should be the signals of putative palmitoylated peptides, which could reduce spectral complexity and achieve double verification for the putative palmitoylated peptides. Finally, the palmitoylome in hepatocellular carcinoma (HCC) cells with different metastasis potentials (MHCC-97L and HCC-LM3 cells) were analyzed for the first time. Totally, 151 proteins were found to be differentially palmitoylated with high confidence, including many important proteins involved in a variety of biological processes, such as protein palmitoylation, cell proliferation, signal transduction, regulation of cell migration, and so on.