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. Cl...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 a-casein as test sample. When the loading amount was 573 pmol/mg matrix, up to 20 a-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 multiphosphopepides 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.展开更多
基金Supported by the National Natural Science Foundation of China（Nos.21105100, 21105007, 21475129）, the Dalian Science and Technology Project, China（No.2012E15SF145） and the Natural Science Foundation of Liaoning Province, China （No.201202054）.
文摘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 a-casein as test sample. When the loading amount was 573 pmol/mg matrix, up to 20 a-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 multiphosphopepides 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.
