Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.
Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.
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DOI：
10.1007/s00726-015-2166-z
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发表时间：
2016-04
期刊：
影响因子：
3.5
通讯作者：
Zhou ZS
中科院分区：
文献类型：
--
作者：
Liu S;Moulton KR;Auclair JR;Zhou ZS
常见但经常被忽视的是，肽基天冬酰胺(AsN或N)的脱酰胺反应产生天冬氨酸(Asp或D)或异天冬氨酸(isAsp或isod)。作为一种自发的、非酶的蛋白质翻译后修饰，脱酰胺伪影很容易在样品制备过程中引入，特别是在蛋白质降解过程中，高阶结构被去除。这一人工产物不仅使真正的脱酰胺化分析复杂化，而且还影响到广泛的化学和酶过程；例如，新产生的天冬氨酸和异天冬氨酸残基可能会阻止或引入新的蛋白水解点，还会将一个天冬氨酸多肽转化为影响定量的多个物种。虽然一般蛋白质分解的中性到中等碱性条件有利于脱酰胺化，但中等酸性条件明显减缓了这一过程。与其他常用的内切酶不同，Glu-C在温和的酸性条件下仍保持活性。因此，如本文所展示的，通过在与质谱学兼容的挥发性缓冲液醋酸铵中简单地在pH 4.5下执行Glu-C消化，可以有效地消除蛋白质分解过程中的脱酰胺伪影。此外，在两种pH下观察到几乎相同的序列特异性(碳酸氢铵为8.0)，使Glu-C在pH为4.5时有效。总而言之，这种方法一般适用于蛋白质分析，因为它需要最少的样品准备，并且使用容易获得的Glu-C蛋白酶。
Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly acid conditions. As such, as demonstrated herein, deamidation artifact during proteolysis was effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH’s (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.