比較不同分離策略搭配液相層析串聯式質譜儀之磷酸化蛋白體學研究
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2016
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磷酸化蛋白體學為近年來許多生化分析學家致力研究的領域，原因是蛋白質的磷酸化對於調控蛋白質活性、細胞間訊息傳遞等生理機制扮演著極為重要的角色。然而，具有磷酸化修飾的蛋白質僅占總體蛋白質的一部份，經水解後所形成的磷酸化胜肽含量偏低，在一般的質譜鑑定流程中容易被含量較高的一般胜肽遮蔽；為了降低樣品的複雜度，並有效提升磷酸化胜肽的鑑定機率，在本研究中使用了羥基脂肪酸修飾之金屬氧化物層析法 （aliphatic hydroxy acid-modified metal oxide chromatography, HAMMOC），先純化出小鼠巨噬細胞株（RAW 264.7）當中的磷酸化胜肽，再選用三種不同的分離方法（fractionation）進行比較。方法分別為強陽離子交換層析法（strong cation chromatography, SCX）、靜電排斥親水性交互作用層析法（electrostatic repulsion hydrophilic interaction chromatography, ERLIC）及液相等電點聚焦分離法（solution isoelectric focusing electrophoresis, sIEF），最後將經三種不同方法分離出來的磷酸化胜肽送入液相層析串聯式質譜儀（LC-MS/MS）進行分析。在蛋白質樣品起始量皆為1毫克的情況下，鑑定到的磷酸化胜肽數目分別如下： SCX-LC-MS/MS 可鑑定到 4336 條磷酸化胜肽、ERLIC-LC-MS/MS為 2064 條磷酸化胜肽，sIEF-LC-MS/MS 則是 2424 條磷酸化胜肽。三種方法總共鑑定到5744條不重複的磷酸化胜肽及 2159 個磷酸化蛋白質，且僅在單一方法中單獨出現的磷酸化胜肽個數依序為 SCX （2430）、ERLIC （438）、sIEF （751）。由此可知，本研究中所選用的三種分離方法具有良好的互補性，能有效提升對磷酸化胜肽的鑑定能力，以期應用於分析磷酸化蛋白體學的研究中。
Phosphorylation is one of the major post-translational modifications that plays a significant role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because its low abundance and the analysis can be hindered by high abundant non-phosphoproteins. In order to improve the identification efficiency of phosphopeptides and reduce the complexity of sample, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) is applied for phosphopeptides enrichment from RAW 264.7 cell lysates. Moreover, strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC) and solution isoelectric focusing (sIEF) were investigated for phosphopeptides fractionation strategies following by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Total 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from 1 mg RAW 264.7 cell lysates combining three fractionation approach. Besides, 4336, 2064 and 2424 phosphopeptides were identified by SCX-LC-MS/MS, ERLIC-LC-MS/MS and sIEF-LC/MS-MS, including 2430, 438 and 751 phosphopeptides only found in SCX, ERLIC and sIEF fractionations specifically. In conclusion, the strategies of three different fractionations have great orthogonality, which improve identification efficiency of phosphopeptides and be suitable for in-depth phosphoproteomic research.
Phosphorylation is one of the major post-translational modifications that plays a significant role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because its low abundance and the analysis can be hindered by high abundant non-phosphoproteins. In order to improve the identification efficiency of phosphopeptides and reduce the complexity of sample, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) is applied for phosphopeptides enrichment from RAW 264.7 cell lysates. Moreover, strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC) and solution isoelectric focusing (sIEF) were investigated for phosphopeptides fractionation strategies following by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Total 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from 1 mg RAW 264.7 cell lysates combining three fractionation approach. Besides, 4336, 2064 and 2424 phosphopeptides were identified by SCX-LC-MS/MS, ERLIC-LC-MS/MS and sIEF-LC/MS-MS, including 2430, 438 and 751 phosphopeptides only found in SCX, ERLIC and sIEF fractionations specifically. In conclusion, the strategies of three different fractionations have great orthogonality, which improve identification efficiency of phosphopeptides and be suitable for in-depth phosphoproteomic research.
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磷酸化胜肽, 羥基脂肪酸修飾之金屬氧化物層析法, 強陽離子交換層析法, 靜電排斥親水交互作用層析法, 液相等電點聚焦分離法, 液相層析串聯式質譜儀, phosphopeptide, HAMMOC, fractionation, SCX, ERLIC, sIEF, LC-MS/MS