Sequential Phosphoproteomic Enrichment through Complementary Metal-Directed Immobilized Metal Ion Affinity Chromatography

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• 作者：Chia-Feng Tsai ; Chuan-Chih Hsu ; Jo-Nan Hung ; Yi-Ting Wang ; Wai-Kok Choong ; Ming-Yao Zeng ; Pei-Yi Lin ; Ruo-Wei Hong ; Ting-Yi Sung ; Yu-Ju Chen
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：January 7, 2014
• 年：2014
• 卷：86
• 期：1
• 页码：685-693
• 全文大小：388K
• ISSN：1520-6882

文摘

Methodologies to enrich heterogeneous types of phosphopeptides are critical for comprehensive mapping of the under-explored phosphoproteome. Taking advantage of the distinct binding affinities of Ga³⁺ and Fe³⁺ for phosphopeptides, we designed a metal-directed immobilized metal ion affinity chromatography for the sequential enrichment of phosphopeptides. In Raji B cells, the sequential Ga³⁺-Fe³⁺-immobilized metal affinity chromatography (IMAC) strategy displayed a 1.5鈥?.5-fold superior phosphoproteomic coverage compared to single IMAC (Fe³⁺, Ti⁴⁺, Ga³⁺, and Al³⁺). In addition, up to 92% of the 6283 phosphopeptides were uniquely enriched in either the first Ga³⁺-IMAC (41%) or second Fe³⁺-IMAC (51%). The complementary properties of Ga³⁺ and Fe³⁺ were further demonstrated through the exclusive enrichment of almost all of 1214 multiply phosphorylated peptides (99.4%) in the Ga³⁺-IMAC, whereas only 10% of 5069 monophosphorylated phosphopeptides were commonly enriched in both fractions. The application of sequential Ga³⁺-Fe³⁺-IMAC to human lung cancer tissue allowed the identification of 2560 unique phosphopeptides with only 8% overlap. In addition to the above-mentioned mono- and multiply phosphorylated peptides, this fractionation ability was also demonstrated on the basic and acidic phosphopeptides: acidophilic phosphorylation sites were predominately enriched in the first Ga³⁺-IMAC (72%), while Pro-directed (85%) and basophilic (79%) phosphorylation sites were enriched in the second Fe³⁺-IMAC. This strategy provided complementary mapping of different kinase substrates in multiple cellular pathways related to cancer invasion and metastasis of lung cancer. Given the fractionation ability and ease of tip preparation of this Ga³⁺-Fe³⁺-IMAC, we propose that this strategy allows more comprehensive characterization of the phosphoproteome both in vitro and in vivo.