Combining [13C6]-phenylisothiocyanate and the Edman degradation reaction: a possible breakthrough for absolute quantitative proteomics together with protein identification.
Combining [13C6]-phenylisothiocyanate and the Edman degradation reaction: a possible breakthrough for absolute quantitative proteomics together with protein identification.
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DOI：
10.1002/rcm.4372
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发表时间：
2010-01
期刊：
影响因子：
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通讯作者：
T. Oe;Masamitsu Maekawa;R. Satoh;Seon-Hwa Lee;T. Goto
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文献类型：
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作者：
T. Oe;Masamitsu Maekawa;R. Satoh;Seon-Hwa Lee;T. Goto
这份手稿描述了一项初步实验的结果，作为一种新的方法的概念证明，该方法无需使用标准蛋白质即可对蛋白质进行绝对定量。在蛋白质组学领域，绝对定量仍然是一个具有挑战性的问题。因此，我们提出将[(13)C(6)]-苯基异硫氰酸酯(PITC)与Edman降解反应相结合作为可能的突破口。以[(13)C(6)]-苯胺和O，O‘-二-2-吡啶硫代碳酸酯为原料合成了[(13)C(6)]-PITC，并以此为内标制备了[(13)C(6)]-苯硫代海因(PTH)-氨基酸。经Edman降解反应证实，模型蛋白牛血清白蛋白(BSA)以PTH-Asp的形式定量释放N-末端氨基酸。PTH-Asp与[(13)C(6)]-PTH-Asp的标准曲线呈良好的线性关系(r(2)=0.9977)。用标准曲线可将BSA定量为PTH-Asp。此外，残留的Des-Asp(1)-BSA为进一步的蛋白质鉴定提供了足够的信息。
This manuscript describes the results of a preliminary experiment performed as 'proof of concept' of a novel approach to absolute quantitation of proteins without the use of standard proteins. Absolute quantitation remains a challenging issue in the proteomics field. Therefore, we propose a combination of [(13)C(6)]-phenylisothiocyanate (PITC) and the Edman degradation reaction as a possible breakthrough. [(13)C(6)]-PITC was synthesized from [(13)C(6)]-aniline with O,O'-di-2-pyridyl thiocarbonate to prepare [(13)C(6)]-phenylthiohydantoin (PTH)-amino acids as internal standards. Upon the Edman degradation reaction, it has been confirmed that a model protein, bovine serum albumin (BSA), releases the N-terminal amino acid quantitatively as PTH-Asp. The standard curve of PTH-Asp against [(13)C(6)]-PTH-Asp showed good linearity (r(2) = 0.9977). BSA could be quantified as PTH-Asp using the standard curve. In addition, the residual des-Asp(1)-BSA provided sufficient information for further protein identification.