Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes
Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes
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DOI：
10.1021/pr070233q
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发表时间：
2007-09-01
影响因子：
4.4
通讯作者：
Hanash, Samir
中科院分区：
文献类型：
--
作者：
Faca, Vitor;Pitteri, Sharon J.;Hanash, Samir
通过质谱法对血清和血浆蛋白质组进行深入分析面临着巨大的蛋白质丰度动态范围和巨大的复杂性的挑战。通过分级分离降低复杂性以促进低丰度蛋白质的质谱分析是有好处的。然而，分级分离会降低通量，并有可能稀释单个蛋白质或导致其损失。在这里，我们研究了完整蛋白质的广泛分级分离对分析深度的贡献。通过由阴离子交换和反相色谱组成的正交二维系统对去除了丰富蛋白质的合并血清进行分级。将所得蛋白质级分等分；一份通过鸟枪式 LC-MS/MS 进行分析，另一份使用 SDS-PAGE 在三维空间中进一步解析为蛋白质条带。切下各个凝胶条带并进行原位消化和质谱分析。我们证明，基于血清中鉴定的蛋白质总数以及第三维 SDS 凝胶分析后凝胶条带中鉴定的特定蛋白质的各个级分的表示，增加分级分离会导致分析深度增加。实施了基于二维血浆分级分离模式的完整蛋白质分析系统 (IPAS)，以高置信度鉴定了 1662 种蛋白质，并代表了色谱迁移率不同的蛋白质亚型。通过对同一组二维级分的等分试样进行重复分析，实现了分析深度的进一步增加，从而全面鉴定了 2254 种蛋白质。我们得出的结论是，通过消耗丰富的蛋白质，然后进行二维蛋白质分级分离和单个级分的 MS 分析，可以对毫升量的血清或血浆中的蛋白质进行相当深度的分析并检测同工型。
In-depth analysis of the serum and plasma proteomes by mass spectrometry is challenged by the vast dynamic range of protein abundance and substantial complexity. There is merit in reducing complexity through fractionation to facilitate mass spectrometry analysis of low-abundance proteins. However, fractionation reduces throughput and has the potential of diluting individual proteins or inducing their loss. Here, we have investigated the contribution of extensive fractionation of intact proteins to depth of analysis. Pooled serum depleted of abundant proteins was fractionated by an orthogonal two-dimensional system consisting of anion-exchange and reversed-phase chromatography. The resulting protein fractions were aliquotted; one aliquot was analyzed by shotgun LC-MS/MS, and another was further resolved into protein bands in a third dimension using SDS-PAGE. Individual gel bands were excised and subjected to in situ digestion and mass spectrometry. We demonstrate that increased fractionation results in increased depth of analysis based on total number of proteins identified in serum and based on representation in individual fractions of specific proteins identified in gel bands following a third-dimension SDS gel analysis. An intact protein analysis system (IPAS) based on a two-dimensional plasma fractionation schema was implemented that resulted in identification of 1662 proteins with high confidence with representation of protein isoforms that differed in their chromatographic mobility. Further increase in depth of analysis was accomplished by repeat analysis of aliquots from the same set of two-dimensional fractions resulting in overall identification of 2254 proteins. We conclude that substantial depth of analysis of proteins from milliliter quantities of serum or plasma and detection of isoforms are achieved with depletion of abundant proteins followed by two-dimensional protein fractionation and MS analysis of individual fractions.