Protein phosphorylation as one of the most important post-translational modifications in mammalian cells regulates numerous biological processes. Here we propose a novel strategy for the selective isolation and sensit...Protein phosphorylation as one of the most important post-translational modifications in mammalian cells regulates numerous biological processes. Here we propose a novel strategy for the selective isolation and sensitive analysis of mul- ti-phosphopeptides based on TiO2-gratfed mesoporous materials, in which MCM-41 and SBA-15 were chosen as the hard templates. The commercialized IMAC and TiO2 nanopartices were further investigated in the phosphopeptide analysis for comparison. The enrichment efficiency was evaluated and measured by MALDI-TOF mass spectrometry. The results indicated that both TiO2-SBA-15 and TiO2-MCM-41 exhibited the preferential affinity to multi-phosphopeptides compared with the other two widely used strategies. The mesoporous TiO2 based protocol showed highly selective and sensitive properties, where phosphopepddes could be identified at femtomole.展开更多
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 Basic Research Program of Chi-na (2007CB714506)the National Natural Science Foundation of China (20735005 & 20925517)+1 种基金Science and Technology Committee of Shanghai (10XD1406000 & 09JC1402600)Shanghai Leading Academic Disci-pline B109
文摘Protein phosphorylation as one of the most important post-translational modifications in mammalian cells regulates numerous biological processes. Here we propose a novel strategy for the selective isolation and sensitive analysis of mul- ti-phosphopeptides based on TiO2-gratfed mesoporous materials, in which MCM-41 and SBA-15 were chosen as the hard templates. The commercialized IMAC and TiO2 nanopartices were further investigated in the phosphopeptide analysis for comparison. The enrichment efficiency was evaluated and measured by MALDI-TOF mass spectrometry. The results indicated that both TiO2-SBA-15 and TiO2-MCM-41 exhibited the preferential affinity to multi-phosphopeptides compared with the other two widely used strategies. The mesoporous TiO2 based protocol showed highly selective and sensitive properties, where phosphopepddes could be identified at femtomole.
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.
