脂族羥基酸修飾的金屬氧化物層析法(HAMMOC)搭配液相層析串聯式質譜分析技術(LC-MS/MS)之磷酸化蛋白質體研究

Abstract

蛋白質磷酸化是一種主要的後轉譯修飾，它調節許多生物系統，如基因表現、訊號傳遞與代謝路徑。但磷酸化修飾為可逆反應且含量低，故需要高靈敏度與具特異性的分析方法。本研究結合脂族羥基酸修飾的金屬氧化物層析法(HAMMOC)與液相層析串聯式質譜分析技術(LC-MS/MS)研究磷酸化蛋白質體學。首先選用丙酮沉澱法與離心過濾法，以比較不同去鹽方法之磷酸化研究；接著於HAMMOC純化後，以等電點聚焦電泳(sIEF)、強陽離子交換層析法(SCX)與親水性層析法(HILIC)降低樣品複雜度，並比較不同分離方法之磷酸化研究；最後，採用無標記定量方法(label-free)進行磷酸化蛋白質體定量。取小鼠巨噬細胞株(RAW 264.7)裂解液，經離心過濾法以及丙酮沉澱法去鹽，毫克含量的蛋白質水解後搭配HAMMOC-sIEF與質譜分析，在兩種方法共鑑定到1231個磷酸化蛋白質與2953段磷酸化胜肽。以離心過濾法搭配HAMMOC與三種分離方法，結果顯示單一HAMMOC-SCX可以鑑定到1747段磷酸化胜肽，再搭配HAMMOC-sIEF和HAMMOC-HILIC可額外鑑定1517段(86.8%)磷酸化胜肽，由此可知三種分離方法具良好的互補性。從小鼠巨噬細胞株之控制組與脂多醣刺激組取200微克的蛋白質，以HAMMOC與LC-MS/MS利用無標記定量方法進行相對定量分析，從中挑選出114個差異表現的磷酸化蛋白質。以上結果顯示選用HAMMOC搭配LC-MS/MS適合深入研究磷酸化蛋白質體學的定性和定量分析。

Protein phosphorylation is one of major post-translational modifications, which regulates many biological intracellular systems, including gene expression, signal transduction pathways and metabolic processes. Phosphorylation, a transient modification and low abundance, requires highly sensitive and specific strategies for its analysis. The combination of aliphatic hydroxyl acid-modified metal oxide chromatography (HAMMOC) enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis were employed for the phosphoproteome analysis in this study. Two desalting methods, including acetone precipitation and centrifugal filter, were used separately before HAMMOC treatment. Moreover, three fractionation strategies, solution isoelectric focusing electrophoresis (sIEF), strong cation exchange chromatography (SCX) and hydrophilic interaction liquid chromatography (HILIC), were utilized to reduce the sample complexity after HAMMOC enrichment. Finally, label-free strategy was implemented for the phosphoproteomic quantification. A total of 2953 phosphopeptides and 1231 phosphoproteins were found in milligram proteins of RAW264.7 cell lysate by combining two desalting methods coupled with HAMMOC-sIEF approach. The use of centrifugal filter and HAMMOC coupled with three various fractionation strategies had shown good complementarity (86.8% more phosphopeptide identifications than SCX only). One hundred and fourteen phosphoproteins were found differentially expressed between control and lipopolysaccharide-treated RAW264.7 from 200 microgram proteins by using label-free comparative approach after HAMMOC and LC-MS/MS. These results indicate that HAMMOC followed by LC-MS/MS analysis is suited for the in-depth analysis of qualitative and quantitative phosphoproteome profiling.