Fränzel Benjamin, Fischer Frank, Steegborn Clemens, Wolters Dirk Andreas
Department of Analytical Chemistry, Biomolecular Mass Spectrometry, Institute of Chemistry and Biochemistry, Ruhr-Universität Bochum, Bochum, Germany.
Proteomics. 2015 Jan;15(1):44-7. doi: 10.1002/pmic.201400015. Epub 2014 Dec 11.
Acetylation is a common PTM of proteins but is still challenging to analyze. Only few acetylome studies have been performed to tackle this issue. Yet, the detection of acetylated proteins in complex cell lysates remains to be improved. Here, we present a proteomic approach with proteinase K as a suitable protease to identify acetylated peptides quantitatively. We first optimized the digestion conditions using an artificial system of purified bovine histones to find the optimal protease. Subsequently, the capability of proteinase K was demonstrated in complex HEK293 cell lysates. Finally, SILAC in combination with MudPIT was used to show that quantification with proteinase K is possible. In this study, we identified a sheer number of 557 unique acetylated peptides originating from 633 acetylation sites.
乙酰化是蛋白质常见的一种翻译后修饰（PTM），但对其进行分析仍具有挑战性。目前仅有少数乙酰化蛋白质组研究致力于解决这一问题。然而，复杂细胞裂解物中乙酰化蛋白质的检测仍有待改进。在此，我们提出一种蛋白质组学方法，使用蛋白酶K作为合适的蛋白酶来定量鉴定乙酰化肽段。我们首先使用纯化的牛组蛋白人工系统优化消化条件以找到最佳蛋白酶。随后，在复杂的HEK293细胞裂解物中证明了蛋白酶K的能力。最后，结合稳定同位素标记氨基酸在细胞培养物中的代谢标记（SILAC）与多维蛋白质鉴定技术（MudPIT）表明使用蛋白酶K进行定量是可行的。在本研究中，我们鉴定出了来自633个乙酰化位点的多达557个独特的乙酰化肽段。
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