Nielsen Michael L, Savitski Mikhail M, Zubarev Roman A
Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, Box 583, Uppsala S-751 23, Sweden.
Mol Cell Proteomics. 2006 Dec;5(12):2384-91. doi: 10.1074/mcp.M600248-MCP200. Epub 2006 Oct 2.
The complexity of the human proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8-12 modified peptides per each unmodified tryptic peptide present at >or=1% level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach.
人类蛋白质组的复杂性在生物体水平上就已十分巨大，在蛋白质组分析过程中，由于体外样品的变化，其复杂性进一步增加。大多数体外变化在体内也会作为翻译后修饰出现。这两种修饰类型只能在事后区分，而无法在分析过程中区分，因此有必要对样品中的每个分子进行分析。通过将新的软件工具ModifiComb应用于MS/MS数据，在代表人类细胞完整蛋白质组的胰蛋白酶混合物中测量了修饰程度。估计每100个未修饰的胰蛋白酶肽中，有8 - 12个修饰肽的含量≥1%，平均每个氨基酸接近一个修饰。这一修饰率高于此前的认知，给分析技术带来了额外挑战。解决该问题的方法在于改进样品制备流程，为数据库搜索引入动态范围调整阈值，使用具有高质量精度和互补碎裂技术的更特异的MS/MS分析，以及通过仅由不常见肽段鉴定额外蛋白质来揭示肽家族。分析前进行广泛的蛋白质分离可降低对串联质谱仪速度和动态范围的要求，并且可能是鸟枪法的可行替代方法。
