Enhanced Multi-phosphopeptide Enrichment and Nano LC-ESI-qTOE-MS Detection Strategy Using Click OEG-CD Matrix
Enhanced Multi-phosphopeptide Enrichment and Nano LC-ESI-qTOE-MS Detection Strategy Using Click OEG-CD Matrix
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使用 Click OEG-CD 矩阵增强多磷酸肽富集和纳米 LC-ESI-qTOE-MS 检测策略
DOI：
10.1007/s40242-014-4179-7
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Liang Xinmiao
中科院分区：
文献类型：
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作者：
Zhao Yanyan;Wang Liheng;Guo Zhimou;Chi Xiaofei;Ma Xiaochi;Qi Yan;Fang Shinong;Li Xiuling;Liang Xinmiao
由于蛋白质消化产物中多磷酸肽的丰度较低，且高丰度磷酸肽的存在会产生离子抑制效应，因此难以对多磷酸肽进行检测和分析。Click OEG-CD[olio（ethylene glycol）（OEG）linkedβ-环糊精（CD）]基质在磷酸肽富集中显示出优异的性能。然而，以前很少检测到多磷酸肽。本研究通过优化每mg基质上样量，提高了多磷酸肽的富集选择性和质谱检测能力。采用纳米液相色谱-电喷雾电离-四极杆飞行时间质谱（LC-ESI-qTOF-MS）进行质谱分析。以单磷酸肽、双磷酸肽和非磷酸肽的混合物为探针，随着每毫克基质上样量的增加，双磷酸肽的富集选择性增强。以典型酶解产物α-酪蛋白为供试品，在优化的上样量条件下，多磷酸肽富集选择性提高。当上样量为573 pmol/mg基质时，可检测到20个α-酪蛋白磷酸肽信号（包括15个多磷酸肽）。结果比我们上次报告的结果好得多。高丰度的非磷酸肽或单磷酸肽的存在以及多磷酸肽与基质之间较强的相互作用降低了离子抑制效应。该研究为Click OEG-CD基质在后续研究中更好地应用于复杂生物样品中多磷酸肽的富集奠定了基础。
Detection and determination of multi-phosphopeptides from protein digestion products are difficult due to the low abundance and ion-suppression effect arised from the existence of their high-abundance counterparts. Click OEG-CD[olio(ethylene glycol)(OEG) linkedβ-cyclodextrin(CD)] matrix has shown excellent performance in phosphopeptide enrichment. However, few multi-phosphopeptides were detected previously. In this investigation, an improved method aiming at enhancing the enrichment selectivity and mass spectrometry(MS) detection of multi-phosphopeptide was developedviaoptimizing the sample loading amount on per mg of matrix. Mass spectra were obtained on a Nano liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (LC-ESI-qTOF-MS). The enrichment selectivity of double-phosphopeptide could be enhanced with the increase of loading amount on per mg of matrix when taking the mixture of mono-, double-, and non-phosphopeptides as probe. Furthermore, the multi-phosphopeptide enrichment selectivity was enhanced under the condition of optimized loading amount when taking the product of typtic digestionα-casein as test sample. When the loading amount was 573 pmol/mg matrix, up to 20α-casein phosphopeptide signals(including 15 multi-phosphopeptides) were detected. The result is much better than that of our previous report. The reduction of ion-suppression effect arised from the existence of high-abundance non- or mono-phosphopeptides and the stronger interactions between multi-phosphopepides and the matrix were contributed to the result. The study could be helpful to the better utilization of Click OEG-CD matrix in the enrichment of multi-phosphopeptide from complex biosamples in the subsequent investigation.