Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications
Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications
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DOI：
10.1074/mcp.t600060-mcp200
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发表时间：
2007-06-01
影响因子：
7
通讯作者：
Ishihama, Yasushi
中科院分区：
文献类型：
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作者：
Sugiyama, Naoyuki;Masuda, Takeshi;Ishihama, Yasushi
我们开发了新的方法，磷酸肽富集脂肪族羟基酸修饰的金属氧化物色谱（MOC）。二氧化钛和氧化锆被成功地应用于在脂肪族羟基酸（如乳酸和β-羟基丙酸）的帮助下富集磷酸肽，以减少酸性非磷酸肽与金属氧化物之间的相互作用。这些方法从磷蛋白标准品中去除了绝大多数非磷酸肽，并且可以容易地鉴定出大量的磷酸肽。该方法与nano-LC-MS/MS系统结合起来没有困难。MOC中磷酸肽的回收率因肽而异，范围从几个百分比到100%，平均几乎为50%。重复性和线性符合要求。在HeLa细胞的细胞质部分的检查中，分别使用乳酸改性的二氧化钛MOC和β-羟基丙酸改性的氧化锆MOC鉴定了超过1000种磷酸肽。这两种方法富集的磷酸肽之间的重叠为40%，合并的结果提供了1646个独特的磷酸肽。据我们所知，这是第一次成功应用一个单一的MOC为基础的方法，磷酸肽富集复杂的生物样品，如细胞裂解物。
We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and beta-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and beta-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates.