利用修飾二氧化鈦之奈米鑽石分析磷酸化胜肽

Abstract

現今利用質譜來分析磷酸化蛋白質或胜肽仍然存在著困難度，所以市面上有很多商業化產品(例如: IMAC、TiO2)，能夠在質譜分析前，將磷酸化蛋白質及磷酸化胜肽做一個濃縮萃取的前置效果，以利於後面質譜鑑定磷酸化位置的便利。 本文以上述目標為方針，利用sol-gel方式合成具功能性的奈米粒子，即在直徑為100奈米的鑽石表面修飾二氧化鈦(TiO2)，藉由其表面對磷酸基團有很好的親合性鍵結，成為具有選擇性的濃縮萃取磷酸化蛋白質及磷酸化胜肽的功能性探針。為了證明此功能性粒子對於磷酸化胜肽有高選擇性和專一性，我們在經過蛋白酶切割作用後的β-casein胜肽溶液中加入20 μg的功能性探針粒子作濃縮萃取實驗；其結果顯示:表面修飾二氧化鈦的奈米鑽石粒子(ND-TiO2)不但對於磷酸化胜肽有很好的專一選擇性，也能在極稀薄的溶液中抓取到磷酸化胜肽(10 fmol)；且對於像HeLa cell lysate的真實樣品也能大比例的萃取其中的磷酸化胜肽。 由實驗結果我們證明了表面修飾二氧化鈦(TiO2)的奈米鑽石粒子對磷酸化胜肽的高度專一性、選擇性及靈敏性。此發現對研究磷酸化蛋白的科學家而言，不但多了一個新的濃縮萃取策略，也希望能夠更廣泛運用在磷酸化蛋白質體學上。

Protein phosphorylation is an important post-translational control of protein activity in cells. However, low abundances, low stoichiometry, and poor ionization of phosphopeptides make the isolation and concentration steps indispensable prior to MS analysis. In this study, we utilized a new probe of high affinity for phosphopeptide enrichment with titanium dioxide-coated nanodiamonds (TiO2-coated NDs). Nanodiamond (ND) holds several unique properties such as small particle size, large specific surface area, wide optical transparency range, and facile surface functionalizability, making it a promising solid-phase substrate for affinity purification mass spectrometry. The enrichment conditions were optimized using tryptic digests of β-casein, and the high specificity of the TiO2-coateed NDs was demonstrated by selectively enriching phosphopeptides from the tryptic digests of protein mixture of β-casein and bovine serum albumin (BSA) with a molar ratio of 1: 5000, followed by MALDI-TOF MS characterization. The new protocol was also coupled with nano-LC-MS/MS system without difficulty. Analysis of tryptic digests from cytoplasmic fraction of HeLa cells yielded numbers of phosphopeptide identifications comparable to that obtained using commercial phosphopeptide isolation tool (Phos-trapTM 96 Enrichment Kit) and almost 59.3% recovery of phosphopeptides, presumable due to the presence of a large numbers of sites available on TiO2-coated NDs for binding or incomplete removal of nonspecific bound peptides. In 120 μg of equivalent of HeLa cell lysates, we identified 696 unique phosphopeptides and 925 phosphorylation sites, indicating the excellent performance of the TiO2-coated NDs.