Complications in the assignment of 14 and 28 Da mass shift detected by mass spectrometry as in vivo methylation from endogenous proteins
Complications in the assignment of 14 and 28 Da mass shift detected by mass spectrometry as in vivo methylation from endogenous proteins
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DOI：
10.1021/ac7021025
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发表时间：
2008-03-01
影响因子：
7.4
通讯作者：
Qin, Jun
中科院分区：
文献类型：
--
作者：
Jung, Sung Yun;Li, Yehua;Qin, Jun
蛋白质甲基化位点的鉴定通常始于对目标蛋白质的蛋白水解酶的MS/MS图谱的数据库搜索，允许在所选的可甲基化的氨基酸残基中添加14和28 Da。尽管我们对赖氨酸和精氨酸甲基化的了解取得了进展，但其他氨基酸残基上的蛋白质甲基化的底物和功能仍然未知。在这里，我们报告了对P53、SMC3、NOS和MeCP2蛋白甲基化的分析。我们发现，大量的多肽可以被修饰在赖氨酸、精氨酸、组氨酸和谷氨酸残基上，质量增加14或28Da，与甲基化一致。令人惊讶的是，当细胞与同位素标记的蛋氨酸培养时，其中大多数没有表现出相应的质量位移，蛋氨酸是合成S-腺苷-L-麦硫氨酸的前体，而腺苷是蛋白质甲基化最常用的甲基供体。这些结果表明，在样品处理和质谱学处理过程中，可能存在外源蛋白质甲基化，或者存在与SAM无关的蛋白质甲基化途径。我们的研究发现，在分离的内源性蛋白质中，蛋白质甲基化的发生率很高，并确定了体内甲基化等修饰的并发症。这项研究为仅仅依靠质量位移进行蛋白质甲基化的质谱学鉴定提供了一个警示，并强调了体内同位素标记作为必要的验证方法的重要性。
Identification of protein methylation sites typically starts with database searching of MS/MS spectra of proteolytic digest of the target protein by allowing addition of 14 and 28 Da in the selected amino acid residues that can be methylated. Despite the progress in our understanding of lysine and arginine methylation, substrates and functions of protein methylation at other amino acid residues remain unknown. Here we report the analysis of protein methylation for p53, SMC3, NOS, and MeCP2. We found that a large number of peptides can be modified on the lysine, arginine, histidine, and glutamic acid residues with a mass increase of 14 or 28 Da, consistent with methylation. Surprisingly, a majority of which did not demonstrate a corresponding mass shift when cells were cultured with isotope-labeled methionine, a precursor for the synthesis of S-adenosyl-L-medhionine (SAM), which is the most commonly used methyl donor for protein methylation. These results suggest the possibility of either exogenous protein methylation during sample handling and processing for mass spectrometry or the existence of SAM-independent pathways for protein methylation. Our study found a high occurrence of protein methylation from SDS-PAGE isolated endogenous proteins and identified complications for assigning such modifications as in vivo methylation. This study provides a cautionary note for solely relying on mass shift for mass spectrometric identification of protein methylation and highlights the importance of in vivo isotope labeling as a necessary validation method.