Cerium ion-chelated magnetic silica microspheres for enrichment and direct determination of phosphopeptides by matrix-assisted laser desorption ionization mass spectrometry

In this study, we employed, for the first time, the Ce4+-chelated magnetic silica microspheres to selectively concentrate phosphopeptides from protein digest products. Cerium ions were chelated onto magnetic silica microspheres using the strategy we established before. After enrichment, the phosphopepticle-conjugated magnetic microspheres were separated from the sample solution just by using a magnet. With the optimized enrichment conditions, the performance of the Ce4+-chelated magnetic microspheres was compared with the Fe3+-chelated microspheres using tryptic digested peptides originating from ovalbumin, a five protein mixture containing phosphoproteins and nonphosphoproteins, as well as a mixture of beta-casein and BSA with a molar ratio of 1:50. Compared to Fe3+, Ce4+-chelated magnetic microspheres exhibited more selective isolation ability for concentrating phosphopeptides from complex mixtures. Even when the amount of the tryptic digest product of BSA is 50 times higher than that of beta-casein in the sample solution, the trace phosphopeptides derived from P-casein can still be concentrated effectively by the Ce4+-chelated magnetic microspheres in only 30 s. Furthermore, we initially utilized the Ce4+-chelated magnetic microspheres to directly enrich phosphopeptides from human serum without extra purification steps or tedious treatment, which opens up a possibility for their further application in phosphoproteomics.