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A high confidence, manually validated human blood plasma protein reference set

机译：高可信度，手动验证的人类血浆蛋白参考集

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Background The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list, HUPO later re-analysed their own original dataset with a more stringent statistical treatment that resulted in a much reduced list of high confidence (at least 95%) proteins compared with their original findings. In order to facilitate the discovery of novel biomarkers in the future and to realize the full diagnostic potential of blood plasma, we feel that there is still a need for an ultra-high confidence reference list (at least 99% confidence) of blood plasma proteins. Methods To address the complexity and dynamic protein concentration range of the plasma proteome, we employed a linear ion-trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) for mass spectrometry (MS) analysis. Both instruments allow the measurement of peptide masses in the low ppm range. Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two consecutive stages of tandem mass spectrometry (MS3). The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved. Results Herein we established a high confidence set of 697 blood plasma proteins and achieved a high 'average sequence coverage' of more than 14 peptides per protein and a median of 6 peptides per protein. All proteins annotated as belonging to the immunoglobulin family as well as all hypothetical proteins whose peptides completely matched immunoglobulin sequences were excluded from this protein list. We also compared the results of using two high-end MS instruments as well as the use of various peptide and protein separation approaches. Furthermore, we characterized the plasma proteins using cellular localization information, as well as comparing our list of proteins to data from other sources, including the HUPO PPP dataset. Conclusion Superior instrumentation combined with rigorous validation criteria gave rise to a set of 697 plasma proteins in which we have very high confidence, demonstrated by an exceptionally low false peptide identification rate of 0.29%.
机译：背景技术人类血浆的巨大诊断潜力引起了人们对其目录编目的极大兴趣和努力，例如人类蛋白质组学组织（HUPO）血浆蛋白质组计划（PPP）试点项目。由于在获取可靠的血浆蛋白列表方面存在挑战，因此HUPO随后通过更严格的统计处理重新分析了自己的原始数据集，与原始结果相比，高置信度（至少95％）蛋白的列表大大减少了。为了促进将来发现新的生物标志物并实现血浆的全部诊断潜力，我们感到仍然需要血浆蛋白的超高可信度参考列表（至少99％的可信度） 。方法为了解决血浆蛋白质组的复杂性和动态蛋白浓度范围，我们采用了线性离子阱-傅里叶变换（LTQ-FT）和线性离子阱-轨道仪（LTQ-Orbitrap）进行质谱（MS）分析。两种仪器都可以测量低ppm范围内的肽质量。此外，我们采用了一个统计评分，该评分允许使用两个连续阶段的串联质谱（MS3）的乘积搜索数据库肽段。母体肽中具有极高质量准确度的MS3的组合可比通常实现的可信度高几个数量级的肽鉴定。结果在本文中，我们建立了697种血浆蛋白的高置信度集，并实现了每个蛋白超过14个肽和每个蛋白中位数为6个肽的高“平均序列覆盖率”。被注释为属于免疫球蛋白家族的所有蛋白质，以及其肽与免疫球蛋白序列完全匹配的所有假定蛋白质均不包括在该蛋白质列表中。我们还比较了使用两种高端质谱仪以及各种肽和蛋白质分离方法的结果。此外，我们使用细胞定位信息来表征血浆蛋白，并将蛋白质列表与其他来源的数据进行比较，包括HUPO PPP数据集。结论优越的仪器结合严格的验证标准产生了一组697种血浆蛋白，我们具有很高的置信度，这是由异常低的0.29％的假肽鉴定率证明的。

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