Multidimensional Strategy for Sensitive Phosphoproteonnics Incorporating Protein Prefractionation Combined with SIMAC, HILIC, and TiO2 Chromatography Applied to Proximal EGF Signaling
Multidimensional Strategy for Sensitive Phosphoproteonnics Incorporating Protein Prefractionation Combined with SIMAC, HILIC, and TiO2 Chromatography Applied to Proximal EGF Signaling
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DOI：
10.1021/pr200641x
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发表时间：
2011-12-01
影响因子：
4.4
通讯作者：
Larsen, Martin R.
中科院分区：
文献类型：
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作者：
Engholm-Keller, Kasper;Hansen, Thomas Aarup;Larsen, Martin R.
全面富集和分级分离对于获得磷酸蛋白质组的广泛覆盖至关重要。这不可避免地导致样本丢失，因此，磷酸蛋白质组学研究通常仅在高丰度样本上进行。在这里，我们提出了一种全面的磷酸化蛋白质组学策略，应用于来自 EGF 刺激的 HeLa 细胞的 400 μg 蛋白质。使用碳酸钠结合超速离心将蛋白质分离成膜和细胞质部分。使用 IMAC (SIMAC) 连续洗脱，然后对单磷酸化肽和非磷酸化肽进行亲水相互作用液相色谱，以及随后对 HILIC 级分进行二氧化钛色谱，将磷酸肽分离为单磷酸化和多磷酸化池。这一策略促进了超过 4700 个独特磷酸肽的鉴定，而 636 个磷酸位点在短期 EGF 刺激后发生了变化，其中许多磷酸位点以前并不知道参与 EGFR 信号转导。我们进一步比较了三种不同的数据处理程序，发现由于重新校准和过滤的不同实现，它们的肽识别率存在很大差异。手动验证使用 MaxQuant 软件专门识别的低分肽子集发现了很大比例的误报识别。这表明，尽管具有高度准确的前体质量测定，但在磷酸化蛋白质组学研究中不应自动报告具有低碎片离子分数的肽。
Comprehensive enrichment and fractionation is essential to obtain a broad coverage of the phosphoproteome. This inevitably leads to sample loss, and thus, phosphoproteomics studies are usually only performed on highly abundant samples. Here, we present a comprehensive phosphoproteomics strategy applied to 400 mu g of protein from EGF-stimulated HeLa cells. The proteins are separated into membrane and cytoplasmic fractions using sodium carbonate combined with ultracentrifugation. The phosphopeptides were separated into monophosphorylated and multiphosphorylated pools using sequential elution from IMAC (SIMAC) followed by hydrophilic interaction liquid chromatography of the mono- and nonphosphorylated peptides and subsequent titanium dioxide chromatography of the HILIC fractions. This strategy facilitated the identification of >4700 unique phosphopeptides, while 636 phosphosites were changing following short-term EGF stimulation, many of which were not previously known to be involved in EGFR signaling. We further compared three different data processing programs and found large differences in their peptide identification rates due to different implementations of recalibration and filtering. Manually validating a subset of low-scoring peptides exclusively identified using the MaxQuant software revealed a large percentage of false positive identifications. This indicates that, despite having highly accurate precursor mass determination, peptides with low fragment ion scores should not automatically be reported in phosphoproteomics studies.