Phos-tag™ Mass Analytical Kit

用于 MALDI-TOF/MS检测，提高检测灵敏度！

　　用于质谱分析的试剂套装。

　　Phos-tag Mass Analytical Kit 是用于质谱分析的试剂套装，可配套 MALDI-TOF/MS 使用。可检测磷酸化分子- Phos-tag^® 复合物，通常可提高低磷酸化分子的检测灵敏度。

试剂盒内容：

● Phos-tag™ MS-101L  5 mg（［C27H29N6O64Zn2］3+ MW：581.4）

● Phos-tag™ MS-101H 5 mg（［C27H29N6O68Zn2］3+ MW：589.4）

● Phos-tag™ MS-101N 10 mg（［C27H29N6OZn2］3+ MW：584.3）

◆原理：

◆优点、特色：

● CH₃COO- 等价结合在 Phos-tag™ MS-101 上。

● 在溶液中，不含有阴离子的 Phos-tag™ MS-101 带有＋3价。

● 检测前需制备 1 mM 的 Phos-tag™ MS-101L，MS-101H 或者 MS-101N（溶于水）。

◆案例、应用：

【使用例子：检测 Phos-tag™ – 磷酸化 LPA 复合体】

由于正电荷增大磷酸化 LPA 检测灵敏度上升

Phos-tag™ 系列

磷酸化蛋白新方法！

　　Phos-tag™ 是一种能与磷酸离子特异性结合的功能性分子。它可用于磷酸化蛋白的分离（Phos-tag™ Acrylamide）、Western Blot 检测（Phos-tag™ Biotin）、蛋白纯化 （Phos-tag™ Agarose）及质谱分析 MALDI-TOF/MS （Phos-tag™ Mass Analytical Kit）。

◆Phos-tag™ 的基本结构：

特点：

与-2价磷酸根离子的亲和性和选择性高于其它阴离子

在 pH 5-8 的生理环境下生成稳定的复合物

◆原理：

◆相关应用：

◆相关产品：

产品名称	用　　途
Phos-tag™ Acrylamide	分离：SDS – PAGE 分离不同磷酸化水平的蛋白
SuperSep Phos-tag™	分离：预制胶中含有 50 μM Phos-tag™ Acrylamide
Phos-tag™ Biotin	检测：代替 Western Blot 检测中的磷酸化抗体
Phos-tag™ Agarose	纯化：通用柱层析，纯化磷酸化蛋白
Phos-tag™ Mass 　Analytical Kit	分析：用于质谱 MALDI-TOF/MS 分析，提高磷酸化分子的检测灵敏度

phos-tag™ 由日本广岛大学研究生院医齿药学综合研究科医药分子功能科学研究室开发。

更多产品信息，请点击：http://phos-tag.jp

Phos-tag 第6版说明书

Phos-tag系列 ver. 8

Q.     Phos-tag™ Mass 用于实验可以使用多少次？

A.     如果每次用量为 5 μL，至少可以使用 1000 次。

Q.     如何选择使用 Phos-tag™ MS-101L，Phos-tag™ MS-101H 和 Phos-tag™ MS-101N ？

A.     Phos-tag™ 101N 含有自然存在的 Zn，101L 与 101H 分别含有 Zn 的同位素 ⁶⁴Zn 和 ⁶⁸Zn。

　    请参考以下建议：

　    摸索条件时使用 101N，其中含有多种同位素，结果比较详细；

　    鉴定磷酸基团是否存在，使用 101L 和 101H，这些试剂分别包含 ⁶⁴Zn 和 ⁶⁸Zn。使用这些试剂检测同一个样品时会产生不同的荷质比。

Q.     如果想测定经过 Phos-tag™ SDS-PAGE 分离得到的样品，是否必须要在凝胶消化之前去除 Phos-tag™？

A.     没有必要。SDS-PAGE 结束之后根据一般的凝胶消化方法进行操作即可。

Q.     能否用于 ESI 质谱？

A.     是的，可以使用。请参考下面的文献，这篇报道使用 Phos-tag™ MS-101N 进行 ESI-MS 分析。

　     在实验过程中，使用了中性溶液，若为酸性溶液会导致 Phos-tag ™ 分离。

        【参考文献】 Anal. Chem. (2008), 80, 2531-2538 (MS-101N ESI-MS)

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·  Phosphoinositide kinase signaling controls ER-PM cross-talk[J]. Molecular biology of the cell, 2016, 27(7): 1170-1180，Omnus D J, Manford A G, Bader J M, et al.

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References on Phos-tag™ Chemistry

• Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule, Rapid Communications of Mass Spectrometry, 17, 2075-2081 (2003), H. Takeda, A. Kawasaki, M. Takahashi, A. Yamada, and T. Koike 

• Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc (II) complex, Dalton Transactions, 1189-1193 (2004), E. Kinoshita, M. Takahashi, H. Takeda, M. Shiro, and T. Koike

• Quantitative analysis of lysophosphatidic acid by time-of-flight mass spectrometry using a phosphate capture molecule, Journal of Lipid Research, 45, 2145-2150 (2004), T. Tanaka, H. Tsutsui, K. Hirano, T. Koike, A. Tokumura, and K. Satouchi

•  Production of 1,2-Didocosahexaenoyl Phosphatidylcholine by Bonito Muscle Lysophosphatidylcholine/Transacylase, Journal of Biochemistry,136, 477-483 (2004), K. Hirano, H. Matsui, T. Tanaka, F. Matsuura, K. Satouchi, and T. Koike

• Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins, Journal of Separation Science, 28, 155-162 (2005), E. Kinoshita, A. Yamada, H. Takeda, E. Kinoshita-Kikuta, and T. Koike

• Detection and Quantification of On-Chip Phosphorylated Peptides by Surface Plasmon Resonance Imaging Techniques Using a Phosphate Capture Molecule, Analytical Chemistry, 77, 3979-3985 (2005), K. Inamori, M. Kyo, Y. Nishiya, Y. Inoue, T. Sonoda, E. Kinoshita, T. Koike, and Y. Katayama

• Phosphate-binding tag: A new tool to visualize phosphorylated proteins, Molecular & Cellular Proteomics, 5, 749-757 (2006), E. Kinoshita, E. Kinoshita-Kikuta, K. Takiyama, and T. Koike

• Enrichment of phosphorylated proteins from cell lysate using phosphate-affinity chromatography at physiological pH, Proteomics, 6, 5088-5095 (2006), E. Kinoshita-Kikuta, E. Kinoshita, A. Yamada, M. Endo, and T. Koike

• Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 360, 160-162 (2007), S. Yamada, H. Nakamura, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, and Y. Shiro

• Label-free kinase profiling using phosphate-affinity polyacrylamide gel electrophresis, Molecular & Cellular Proteomics, 6, 356-366 (2007), E. Kinoshita-Kikuta, Y. Aoki, E. Kinoshita, and T. Koike

• A SNP genotyping method using phosphate-affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 361, 294-298 (2007), E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike (The phosphate group at DNA-terminal is efficiently captured by Zn²⁺.Phos-tag.)

• Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule, Journal of Biomolecular Techniques, 18, 278-286 (2007), T. Nakanishi, E. Ando, M. Furuta, E. Kinoshita, E. Kikuta-Kinoshita, T. Koike, S. Tsunasawa, and O. Nishimura

• A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis, Analytical Biochemistry, 378, 102-104 (2008), E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike

• Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate- affinity SDS-PAGE, Proteomics, 8, 2994-3003 (2008), E. Kinoshita, E. Kinoshita-Kikuta, M. Matsubara, S. Yamada, H. Nakamura, Y. Shiro, Y. Aoki, K. Okita, and T. Koike

• FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway, Nature Structural & Molecular Biology, 15, 1138-1146 (2008), M. Ishiai, H. Kitao, A. Smogorzewska, J. Tomida, A. Kinomura, E. Uchida, A. Saberi, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, S. Tashiro, S. J. Elledge, and M. Takata

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• Two-dimensional phosphate affinity gel electrophoresis for the analysis of phosphoprotein isotypes , Electrophoresis, 30, 550-559 (2009), E. Kinoshita, E. Kinoshita-Kikuta, M. Matsubara, Y. Aoki, S. Ohie, Y. Mouri, and T. Koike

• Formation of lysophosphatidic acid, a wound-healing lipid, during digestion of cabbage leaves, Bioscience, Biotechnology, and Biochemistry,73, 1293-300 (2009), T. Tanaka, G. Horiuchi, M. Matsuoka, K. Hirano, A. Tokumura, T. Koike, and K. Satouchi

• A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the dephosphorylation of phosphopeptides, Analytical Biochemistry, 388, 235-241, (2009), K. Takiyama, E. Kinoshita, E. Kinoshita-Kikuta, Y. Fujioka, Y. Kubo, and T. Koike

• Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates, Analytical Biochemistry, 389, 83-85, (2009), E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike

• Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose, Proteomics, 9, 4098- 4101 (2009), E. Kinoshita, E. Kinoshita-Kikuta, H. Ujihara, and T. Koike

• Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE, Nature Protocols, 4, 1513-1521 (2009), E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike

• A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag, Rapid Communications in Mass Spectrometry, 24, 1075-1084 (2010), J. Morishige, M. Urikura, H. Takagi, K. Hirano, T. Koike, T. Tanaka, and K. Satouchi

• Genotyping and mapping assay of single-nucleotide polymorphisms in CYP3A5 using DNA-binding zinc(II) complexes, Clinical Biochemistry, 43, 302-306 (2010), E. Kinoshita, E. Kinoshita-Kikuta, H. Nakashima, and T. Koike

• The DNA-binding activity of mouse DNA methyltransferase 1 is ragulated phosphorylation with casein kinase 1σ/ε, Biochemical Journal, 427, 489-497 (2010), Y. Sugiyama, N. Hatano, N. Sueyoshi, I. Suetake, S. Tajima, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, and I. Kameshita