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.
