Shotgun Protein Sequencing with Meta-contig Assembly
Shotgun Protein Sequencing with Meta-contig Assembly
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DOI：
10.1074/mcp.m111.015768
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发表时间：
2012-10-01
影响因子：
7
通讯作者：
Bandeira, Nuno
中科院分区：
文献类型：
--
作者：
Guthals, Adrian;Clauser, Karl R.;Bandeira, Nuno
对未知蛋白质（例如来自具有未测序基因组的生物体的抗体或蛋白质）的串联质谱（MS/MS）谱进行全长从头测序仍然是一个具有挑战性的开放问题。设计用于对每个 MS/MS 谱图进行单独测序的传统算法受到不完整的肽碎片或低信噪比的限制，并且往往会导致测序精度低的短从头序列。我们开发的鸟枪法蛋白质测序 (SPS) 方法是为了改善这些限制，首先从相同的肽（重叠群）中查找未识别的光谱组，然后为每个组装的光谱集（重叠群序列）导出共有的从头序列。但是，虽然 SPS 能够更准确地重建比从单个 MS/MS 谱图恢复的更长的从头序列，但它仍然需要与同源蛋白质进行容错匹配，以将较小的重叠群序列分组为全长蛋白质序列，从而限制了其对来自注释不良蛋白质的序列的有效性。使用低分辨率和高分辨率 CID 以及高分辨率 HCD MS/MS 谱图，我们通过 Meta-SPS 算法解决了这一限制，该算法旨在重叠并进一步将 SPS 重叠群组装成 Meta-SPS 从头重叠群序列，该序列延伸长达 100 个氨基酸，准确度超过 97%，而无需任何同源蛋白质序列知识。我们使用通过单独的酶消化获得的不同 MS/MS 数据集演示了 Meta-SPS，并讨论了剩余的从头测序限制与 MS/MS 采集设置的关系。分子与细胞蛋白质组学 11：10.1074/mcp.M111.015768, 1084-1096, 2012。
Full-length de novo sequencing from tandem mass (MS/MS) spectra of unknown proteins such as antibodies or proteins from organisms with unsequenced genomes remains a challenging open problem. Conventional algorithms designed to individually sequence each MS/MS spectrum are limited by incomplete peptide fragmentation or low signal to noise ratios and tend to result in short de novo sequences at low sequencing accuracy. Our shotgun protein sequencing (SPS) approach was developed to ameliorate these limitations by first finding groups of unidentified spectra from the same peptides (contigs) and then deriving a consensus de novo sequence for each assembled set of spectra (contig sequences). But whereas SPS enables much more accurate reconstruction of de novo sequences longer than can be recovered from individual MS/MS spectra, it still requires error-tolerant matching to homologous proteins to group smaller contig sequences into full-length protein sequences, thus limiting its effectiveness on sequences from poorly annotated proteins. Using low and high resolution CID and high resolution HCD MS/MS spectra, we address this limitation with a Meta-SPS algorithm designed to overlap and further assemble SPS contigs into Meta-SPS de novo contig sequences extending as long as 100 amino acids at over 97% accuracy without requiring any knowledge of homologous protein sequences. We demonstrate Meta-SPS using distinct MS/MS data sets obtained with separate enzymatic digestions and discuss how the remaining de novo sequencing limitations relate to MS/MS acquisition settings. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.015768, 1084-1096, 2012.