親和層析法（IMAC）搭配質譜技術對LPS-treated RAW 264.7之磷酸蛋白質體學研究
No Thumbnail Available
Date
2012
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
對於生物體而言，胜肽或蛋白質的磷酸化是極其重要的。因蛋白質磷酸化可調節許多生物體內化學反應，如新陳代謝（metabolism）、細胞間的訊號傳遞（signal transduction）等。因此，有效並正確的鑑定磷酸化位置可以幫助開發新的藥物或了解疾病的發生。
在本篇論文中，使用固定金屬離子親和層析法（Immobilized Metal Affinity Chromatography, IMAC）純化磷酸化蛋白質標準品α-酪蛋白（α-casein）之磷酸化胜肽，搭配奈流液相層析串聯質譜（nano-LC ESI MS/MS）可發現其特異性並不亞於市售金屬氧化物親和層析法（Metal Oxide Affinity Chromatography, MOAC）之純化結果。並討論利用LTQ-XL分別使用質譜掃描策略neutral loss MS3（NL-MS3）及multistage activation（MSA）偵測磷酸化胜肽，在一張圖譜中MSA可獲得的胜肽碎片資訊較NL-MS3來的多，當進行資料庫搜尋鑑定胜肽時MSA也可提升其可信度。
在第二部分，會針對以脂多醣體（Lipopolysaccharide, LPS）刺激後的老鼠巨噬細胞株（RAW 264.7），在前處理的部分分別使用Amicon® Ultra、Zeba™ desalting columns及丙酮蛋白質沉澱法三種不同的萃取策略去除界面活性劑及干擾鹽類，接著利用Fe-NTA IMAC純化磷酸化胜肽來進行深入探討，不同的萃取方式所殘留的小分子可能會影響後續純化的效率，實驗結果證實選擇Zeba™ desalting columns作為前處理方法可使IMAC得到最多的磷酸化胜肽/蛋白質。其後，優化了胜肽與IMAC固相載體之最佳使用比例，發現當使用不足或過量的IMAC固相載體會減少磷酸化胜肽的數量或降低其特異性。
Protein phosphorylation plays a significant role in biological process. Since reversible phosphorylation events is known to be involved regulating many intracellular biological processes such as metabolism, signal transduction. The detection and identification of the phosphorylation sites are important to understand physiological states. Tryptic peptides generated from α-casein were enriched phosphopeptides by immobilized metal affinity chromatography (IMAC); different MS acquisition methods using LTQ-XL, neutral loss MS3 (NL-MS3) and multistage activation (MSA) were compared with results based on the number of phosphopeptide identifications. The MSA method performed as well or better then NL-MS3, the spectra represented higher signal intensities from the fragment ions and it is a faster route compared with NL-MS3. The efficiency of phosphopeptide enrichment is severely affected by various reagents commonly utilized in cell biology protocols, it is important to investigate the effect on different pre-purification step prior to phosphopeptide enrichment. Phosphopeptide-enrich strategies were applied to purify LPS-treated RAW 264.7, and different protein extraction methods were also investigated to monitor the influence in downstream enrichment procedures. Zeba desalting columns (Thermo Scientific) was appropriate for pre-purification due to the minimum interference before phosphopeptide enrichment. Furthermore, the peptides-to-IMAC beads ratio was also optimized.
Protein phosphorylation plays a significant role in biological process. Since reversible phosphorylation events is known to be involved regulating many intracellular biological processes such as metabolism, signal transduction. The detection and identification of the phosphorylation sites are important to understand physiological states. Tryptic peptides generated from α-casein were enriched phosphopeptides by immobilized metal affinity chromatography (IMAC); different MS acquisition methods using LTQ-XL, neutral loss MS3 (NL-MS3) and multistage activation (MSA) were compared with results based on the number of phosphopeptide identifications. The MSA method performed as well or better then NL-MS3, the spectra represented higher signal intensities from the fragment ions and it is a faster route compared with NL-MS3. The efficiency of phosphopeptide enrichment is severely affected by various reagents commonly utilized in cell biology protocols, it is important to investigate the effect on different pre-purification step prior to phosphopeptide enrichment. Phosphopeptide-enrich strategies were applied to purify LPS-treated RAW 264.7, and different protein extraction methods were also investigated to monitor the influence in downstream enrichment procedures. Zeba desalting columns (Thermo Scientific) was appropriate for pre-purification due to the minimum interference before phosphopeptide enrichment. Furthermore, the peptides-to-IMAC beads ratio was also optimized.
Description
Keywords
固定金屬離子親和層析法, 磷酸化胜肽, 液相層析串聯質譜儀, IMAC, phosphopeptide, LC-MS/MS