高等学校化学研究 ›› 2015, Vol. 31 ›› Issue (1): 44-52.doi: 10.1007/s40242-014-4179-7
Enhanced Multi-phosphopeptide Enrichment and Nano LC-ESI-qTOF-MS Detection Strategy Using Click OEG-CD Matrix
ZHAO Yanyan1, WANG Liheng2, GUO Zhimou3, CHI Xiaofei2, MA Xiaochi1, QI Yan1, FANG Shinong1, LI Xiuling3, LIANG Xinmiao3
- 1. Pharmacy College, Dalian Medical University, Dalian 116044, P. R. China;
2. The Second Hospital of Dalian City, Dalian 116011, P. R. China;
3. Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, P. R. China
Enhanced Multi-phosphopeptide Enrichment and Nano LC-ESI-qTOF-MS Detection Strategy Using Click OEG-CD Matrix
ZHAO Yanyan1, WANG Liheng2, GUO Zhimou3, CHI Xiaofei2, MA Xiaochi1, QI Yan1, FANG Shinong1, LI Xiuling3, LIANG Xinmiao3
- 1. Pharmacy College, Dalian Medical University, Dalian 116044, P. R. China;
2. The Second Hospital of Dalian City, Dalian 116011, P. R. China;
3. Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, P. R. China
摘要：
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) linked β-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 developed via optimizing 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 digestion α-casein as test sample. When the loading amount was 573 pmol/mg matrix, up to 20 α-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.