In this study, a new strategy named two-step IMAC is demonstated as a novel prelude to MS analysis of phosphoproteome by increasing the enrichment factor of phosphoproteins/phosphopeptides from a pro tein mixture. In this method, the first IMAC was per formed at the protein level to extact the minu te amount of phosphoproteins present in the sample. During this step, nonphosphoproteins and other undesired chemicals or inhibitors were excluded. After tryptic digestion, the second IMAC was per formed at the peptide level to enrich phosphopeptides present in the tryptic digest, and the eluent from the second IMAC was analyzed by MALDI-MS. It is particularly noticeable that the eluent from the first IMAC can be directly digested by trypsin without buffer exchange. Our results revealed that β-ca sein that was spiked in a protein mixture can be success fully extracted by the first IMAC at a concentration of less than 1-3%, and the two phosphopeptides of β-ca sein with single and fourphosphorylation sites, respeclively, canbe captured by the second IMAC. It was found that the two-step IMAC method could significantly reduce non-specific bindings from unwanted proteins and greatly enhance the MALDI-MS signal of phosphopeptide ions compared to the typical one-step IMAC, by which only IMAC at the peptide level was performed. Two-step IMAC was also found to tolerate a greater amount and a greater concentration range of proteins than one-step IMAC, which is especially important when analyzing complicated un known samples. Furthermore, the MS signal of phosphopeptide ions did not appear to be de graded by the presence of biological matrixes, such as the cell lysate in which the β-casein was spiked in.
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