Mesoporous TiO2 Nanowires for Selective and Reproducible Enrichment of Phosphopeptides

[[abstract]]摘要 在磷酸化蛋白質體學的研究中，人們廣泛的使用二氧化鈦粒子為親和性固相萃取材料，用於純化及分離磷酸化的蛋白質片段，以提高質譜儀器分析的靈敏度。儘管目前已經有許多有關二氧化鈦材料的合成方法文獻發表，但是對於二氧化鈦材料的本身的物理特性如、晶體結構、孔洞性質、材料表面積是否影響萃取磷酸化胜肽的選擇性，並沒有被明確的探討，因此我們利用具有孔洞大一致性的二氧化鈦奈米線，進行二氧化鈦材料特性對磷酸化胜肽的選擇性萃取能力的研究。 藉由調配標準品的莫耳數比例 ( β-casein:BSA ) 及真實樣品 ( HeLa cell ) 作為二氧化鈦奈米線的選擇性實驗，其結果顯示二氧化鈦奈米線擁有良好的選擇性 ( 1:1500莫耳數比及平均92.0 % 的磷酸化胜肽選擇率 )。接下來利用HeLa cell ( 200μg ) 進行三次重覆的實驗，證明二氧化鈦奈米線具有63.3 % 的磷酸化胜肽重覆率及71.1 % 的磷酸化蛋白質重覆率，而三次重覆實驗的磷酸化胜肽訊號強度平均相對標準偏差百分比為 20.9 % 。最後利用α-casein作為內標準品，應用在HeLa cell ( 100μg、50μg、10μg ) 微量化的定量上，其平均線性平方達到0.95以上的表現。 因此，我們認為孔徑大小一致的二氧化鈦奈米線，應用於蛋白質體學的定量及磷酸化胜肽的選擇性將有很大的幫助。[[abstract]]Abstract Titanium dioxide (TiO2) affinity enrichment has been widely used in phosphoproteomic studies. Although many attempts have been made to fabricate TiO2-based materials for selective enrichment of phosphopeptides, the effects of structural properties, such as crytsallinity, pore diameter, porosity, and specific surface area of TiO2, on selectivity has not been widely explored. Herein, we synthesized TiO2 nanowires with well-defined nanopores and high specific surface area for selective and reproducible enrichment of phosphopeptides from complex peptide mixture. TiO2 nanowires showed improved performance than TiO2 beads for selective and reproducible detection of phosphopeptides from tryptic digests of β-casein/BSA mixture (1:1500 molar ratio) and HeLa cell lysates (200 μg). We attributed the high selectivity and reproducibility of TiO2 nanowires for phosphopeptide enrichment to the narrow pore diameter distribution and not to the crytsallinity. The average coefficient of variation of phosphopeptide peak intensity values between three replicates was 20.9 %. Venn diagrams showed extensive overlap of identified phosphopeptides (63.3 %) and phosphoproteins (71.1 %) between three replicates. Quantitative phosphopeptide analysis of HeLa cell lysate digest (from 100 μg to 10 μg ) externally spiked α-casein as internal standard reveal good linear relationship with an average correlation coefficient greater than 0.95. Based on these results, we expect that TiO2 nanowires will be of great interest for quantitative phosphoproteome analysis.[[note]]碩