Reliable FASP-based procedures for optimal quantitative proteomic and phosphoproteomic analysis on samples from acute myeloid leukemia patients.
Reliable FASP-based procedures for optimal quantitative proteomic and phosphoproteomic analysis on samples from acute myeloid leukemia patients.
复制标题
DOI：
10.1186/s12575-016-0043-0
复制
发表时间：
2016
影响因子：
6.4
通讯作者：
Selheim F
中科院分区：
文献类型：
--
作者：
Hernandez-Valladares M;Aasebø E;Mjaavatten O;Vaudel M;Bruserud Ø;Berven F;Selheim F
基于质谱分析的令人满意的样品制备是蛋白质组学工作流程中的关键步骤。样品制备的质量和重现性可以决定蛋白质组学结果的覆盖率和可信度。迄今为止，已经描述了几种方法来生产适合质谱分析的肽，然后根据需要制定富集翻译后修饰肽的策略。其中，过滤辅助样品制备（FASP）是一种在胰蛋白酶消化之前去除变性剂、还原剂、烷化剂、脂质和核酸的方法。尽管该方法描述了高蛋白水解消化和污染物去除效率，但过滤器故障和因此导致的样品完全损失可能会阻止蛋白质组学界使用这种方法。根据我们的质量控制判断，我们能够执行可靠且可重复的 FASP 进行质谱分析，从而可以分别从急性髓系白血病 (AML) 患者的 20 μg 和 320 μg 蛋白质裂解液中定量 2141 种蛋白质和 3694 种磷酸肽。使用固定化金属离子亲和层析 (IMAC) 方法可得到特异性富集磷酸肽的样品，除了丰富的单磷酸肽外，还可对大量二磷酸肽和多磷酸肽进行定量。相似数量的定量蛋白质和局部磷酸位点证明了三个生物重复的工作流程的高再现性，并通过其分子功能的相似分布得到证实。我们发现，FASP 程序与 StageTip 混合模式分级分离和 IMAC 的组合分别是 AML 蛋白质组和磷酸蛋白质组的可重复和深入研究的出色工作流程。通过在添加蛋白质样品之前对过滤器质量进行简单的测试，可以在没有过滤器故障的风险的情况下执行 FASP 程序。在此，我们展示了一种高效且可重复的基于 FASP 的管道，用于 AML 患者样本的蛋白质组和磷酸化蛋白质组分析，该管道也可用于任何其他蛋白质样本的分析。本文的在线版本 (doi:10.1186/s12575-016-0043-0) 包含补充材料，可供授权用户使用。
Satisfactory sample preparation for mass spectrometry-based analysis is a critical step in the proteomics workflow. The quality and reproducibility of sample preparation can determine the coverage and confidence of proteomics results. Up to date, several methodologies have been described to produce suitable peptides for mass spectrometry analysis, followed by strategies for enrichment of post-translational modified peptides, if desired. Among them, the filter-aided sample preparation (FASP) has been introduced as a method to allow for removal of denaturants, reductants, alkylators, lipids and nucleic acids prior to trypsin digestion. Despite the high proteolytic digestion and contaminant removal efficiency described for this method, filter failure and consequently complete sample loss can discourage the use of this approach by the proteomic community. As judged by our quality controls, we were able to perform reliable and reproducible FASP for mass spectrometry analysis that allowed the quantification of 2141 proteins and 3694 phosphopeptides from as little as 20 and 320 μg of protein lysate from acute myeloid leukemia (AML) patients, respectively. Using the immobilized metal ion affinity chromatography (IMAC) method resulted in samples specifically enriched in phosphopeptides and allowed the quantification of a high number of both di- and multi-phosphopeptides in addition to the abundant mono-phosphopeptides. The workflows’ high reproducibility from three biological replicates was demonstrated by the similar number of quantified proteins and localized phosphosites, and confirmed by the similar distributions of their molecular functions. We found that the combination of the FASP procedure with StageTip mixed-mode fractionation and IMAC are excellent workflows for the reproducible and deep study of AML proteomes and phosphoproteomes, respectively. The FASP procedure can be carried out without the risk of filter failure by performing a simple test of the filter quality before adding the protein sample. Herein, we demonstrate an efficient and reproducible FASP-based pipeline for the proteomic and phosphoproteomic analysis of AML patient samples which also can be used for the analysis of any other protein samples. The online version of this article (doi:10.1186/s12575-016-0043-0) contains supplementary material, which is available to authorized users.