Successive and selective release of phosphorylated peptides captured by hydroxy acid-modified metal oxide chromatography
Successive and selective release of phosphorylated peptides captured by hydroxy acid-modified metal oxide chromatography
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DOI：
10.1021/pr800305y
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发表时间：
2008-10-01
影响因子：
4.4
通讯作者：
Ishihama, Yasushi
中科院分区：
文献类型：
--
作者：
Kyono, Yutaka;Sugiyama, Naoyuki;Ishihama, Yasushi
我们开发了一种新的方法，通过使用不同的缓冲液从填充了羟基酸修饰的金属氧化物(如钛和氧化锆)的单个微柱中连续洗脱磷酸肽来扩大磷酸蛋白质组的覆盖率。考察了洗脱条件，最大限度地回收了三种标准磷酸蛋白中的磷酸肽。仲胺，如哌啶和吡咯烷，提供了比使用氢氧化铵和磷酸盐缓冲液的传统条件更好的效率。此外，这些仲胺的洗脱提供了在常规条件下不能在HeLa细胞裂解物分析中洗脱的独特的磷肽。在此基础上，我们用5%氢氧化铵溶液、5%哌啶溶液和5%吡咯烷溶液串联洗脱，对用单一金属氧化物微柱捕获的磷酸肽进行了分级。我们从100mg的HeLa细胞中鉴定出1803个非冗余的磷酸肽，与常规条件下的结果相比，磷酸肽的数量增加了1.6倍，总峰面积增加了1.9倍。由于这种方法适用于任何单一的基于TIP的方法，而不与其他富集法相结合，因此这一简单的策略可以很容易地作为一种选择并入现有的磷肽富集法中，并且将适合于以并行格式进行高通量分析。
We developed a novel approach to enlarge phosphoproteome coverage using successive elution of phosphopeptides with various buffers in series from a single microcolumn packed with hydroxy acid-modified metal oxides, such as titanic and zirconia. Elution conditions were investigated to maximize the recovery of phosphopeptides from three standard phosphoproteins. Secondary amines, such as piperidine and pyrrolidine, provided better efficiency than the conventional conditions using ammonium hydroxide and phosphate buffers. Furthermore, elution with these secondary amines provided unique phosphopeptides that were not eluted under the conventional conditions in the analysis of HeLa cell lysates. On the basis of these results, we fractionated phosphopeptides captured by a single metal oxide microcolumn using successive elution with 5% ammonium hydroxide solution, 5% piperidine solution and 5% pyrrolidine solution in series. We identified 1803 nonredundant phosphopeptides from 100 mu g of HeLa cells, which represented a 1.6-fold increase in phosphopeptide number and a 1.9-fold increase in total peak area of phosphopeptides in comparison with the results obtained under the conventional conditions. Since this approach is applicable to any single tip-based protocol without coupling with other enrichment methods, this simple strategy can be easily incorporated as an option into existing protocols for phosphopeptide enrichment, and would be suitable for high-throughput analysis in a parallel format.