Improved titanium dioxide enrichment of phosphopeptides from HeLa cells and high confident phosphopeptide identification by cross-validation of MS/MS and MS/MS/MS spectra
Improved titanium dioxide enrichment of phosphopeptides from HeLa cells and high confident phosphopeptide identification by cross-validation of MS/MS and MS/MS/MS spectra
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DOI：
10.1021/pr070152u
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发表时间：
2007-11-01
影响因子：
4.4
通讯作者：
Veenstra, Timothy D.
中科院分区：
文献类型：
--
作者：
Yu, Li-Rong;Zhu, Zhongyu;Veenstra, Timothy D.
富集对于磷酸化蛋白质组分析是必不可少的，因为磷酸化蛋白质通常以低丰度存在于细胞中。最近，二氧化钛（TiO 2）已被证明可以从简单的肽混合物中富集具有高度特异性的磷酸肽;然而，该技术尚未优化。在本研究中，观察到显着的非特异性结合时，蛋白质组样品被施加到二氧化钛柱。在装载肽混合物后用NH（4）Glu溶液洗涤柱显著提高了TiO 2磷酸肽富集的效率，回收率高达84%。此外，对于蛋白质组样品，独特的磷酸肽鉴定增加了2倍以上。使用NH（4）Glu进行TiO 2柱洗涤不会显著降低磷酸肽回收率。共858磷酸肽对应于1034个不同的磷酸位点已确定从HeLa细胞使用改进的TiO 2富集程序结合数据依赖性中性损失纳米RPLC-MS 2-MS 3分析。分别有41%和35%的磷酸肽仅通过MS 2和MS 3鉴定，而24%的磷酸肽通过MS 2和MS 3两者鉴定。通过MS 2和MS 3扫描对磷酸肽分配进行交叉验证，得到最高的鉴别置信度（99.5%）。在这项研究中发现的许多磷酸化位点似乎是新的，包括抗原Ki-67，核仁磷蛋白p130，和糖浆蛋白的网站。研究还表明，通过反向序列数据库搜索策略评估磷酸肽鉴定的置信水平可能会低估假阳性率。
Enrichment is essential for phosphoproteome analysis because phosphorylated proteins are usually present in cells in low abundance. Recently, titanium dioxide (TiO2) has been demonstrated to enrich phosphopeptides from simple peptide mixtures with high specificity; however, the technology has not been optimized. In the present study, significant non-specific bindings were observed when proteome samples were applied to TiO2 columns. Column wash with an NH(4)Glu solution after loading peptide mixtures significantly increased the efficiency of TiO2 phosphopeptide enrichment with a recovery of up to 84%. Also, for proteome samples, more than a 2-fold increase in unique phosphopeptide identifications has been achieved. The use of NH(4)Glu for a TiO2 column wash does not significantly reduce the phosphopeptide recovery. A total of 858 phosphopeptides corresponding to 1034 distinct phosphosites has been identified from HeLa cells using the improved TiO2 enrichment procedure in combination with data-dependent neutral loss nano-RPLC-MS2-MS3 analysis. While 41 and 35% of the phosphopeptides were identified only by MS2 and MS3, respectively, 24% was identified by both MS2 and MS3. Cross-validation of the phosphopeptide assignment by MS2 and MS3 scans resulted in the highest confidence in identification (99.5%). Many phosphosites identified in this study appear to be novel, including sites from antigen Ki-67, nucleolar phosphoprotein p130, and Treacle protein. The study also indicates that evaluation of confidence levels for phosphopeptide identification via the reversed sequence database searching strategy might underestimate the false positive rate.