De novo sequencing of unique sequence tags for discovery of post-translational modifications of proteins.
De novo sequencing of unique sequence tags for discovery of post-translational modifications of proteins.
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DOI：
10.1021/ac801123p
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发表时间：
2008-10-15
影响因子：
7.4
通讯作者：
Smith, Richard D.
中科院分区：
文献类型：
--
作者：
Shen, Yufeng;Tolic, Nikola;Hixson, Kim K.;Purvine, Samuel O.;Anderson, Gordon A.;Smith, Richard D.
De novo测序是一种质谱分析方法，用于发现蛋白质的翻译后修饰；然而，这种方法仍处于起步阶段，由于其有限的可靠性，仍未广泛应用于蛋白质组学实践。在这项工作中，我们描述了一种基于蛋白质组UStags鉴定的蛋白质修饰发现的从头测序方法。从酵母裂解物的多肽和多肽的傅里叶变换串联质谱数据中获得新生序列信息，并根据过滤水平选择获得的新生序列，以提供有限但高质量的UStags子集。然后将dna预测的数据库蛋白序列与UStags进行比较，并使用UStags之间或内部观察到的差异（即UStags的前缀和后缀序列以及UStags本身）来推断可能的序列修饰。通过这种全新的ustag方法，我们发现了由于氨基酸突变和/或对预测蛋白质序列的多次修改而导致的几种酵母蛋白质序列中的一些意想不到的差异。为了确定错误发现率，我们独立使用了两个随机（错误）数据库进行序列匹配，估计de novo-UStag方法的错误发现率为~ 3%。研究并描述了影响该方法可靠性的因素（如新生测序噪声残馀和冗余序列的存在）和灵敏度。结合de novo-UStag方法通过发现新的蛋白质修饰来补充先前报道的UStag方法。
De novo sequencing is a spectrum analysis approach for mass spectrometry data to discover post-translational modifications in proteins; however, such an approach is still in its infancy and is still not widely applied to proteomic practices due to its limited reliability. In this work, we describe a de novo sequencing approach for the discovery of protein modifications based on identification of the proteome UStags. The de novo information was obtained from Fourier-transform tandem mass spectrometry data for peptides and polypeptides from a yeast lysate, and the de novo sequences obtained were selected based on filter levels designed to provide a limited yet high quality subset of UStags. The DNA-predicted database protein sequences were then compared to the UStags, and the differences observed across or in the UStags (i.e., the UStags’ prefix and suffix sequences and the UStags themselves) were used to infer possible sequence modifications. With this de novo–UStag approach, we uncovered some unexpected variances within several yeast protein sequences due to amino acid mutations and/or multiple modifications to the predicted protein sequences. To determine false discovery rates, two random (false) databases were independently used for sequence matching, and ∼3% false discovery rates were estimated for the de novo–UStag approach. The factors affecting the reliability (e.g., existence of de novo sequencing noise residues and redundant sequences) and the sensitivity of the approach were investigated and described. The combined de novo–UStag approach complements the UStag method previously reported by enabling the discovery of new protein modifications.
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影响因子：
4.4
作者：
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影响因子：
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DOI：
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