Successive and selective release of phosphorylated peptides captured by hydroxy acid-modified metal oxide chromatography

We developed a novel approach to enlarge phosphoproteome coverage using successive elution of phosphopeptides with various buffers in series from a single microcolumn packed with hydroxy acid-modified metal oxides, such as titanic and zirconia. Elution conditions were investigated to maximize the recovery of phosphopeptides from three standard phosphoproteins. Secondary amines, such as piperidine and pyrrolidine, provided better efficiency than the conventional conditions using ammonium hydroxide and phosphate buffers. Furthermore, elution with these secondary amines provided unique phosphopeptides that were not eluted under the conventional conditions in the analysis of HeLa cell lysates. On the basis of these results, we fractionated phosphopeptides captured by a single metal oxide microcolumn using successive elution with 5% ammonium hydroxide solution, 5% piperidine solution and 5% pyrrolidine solution in series. We identified 1803 nonredundant phosphopeptides from 100 mu g of HeLa cells, which represented a 1.6-fold increase in phosphopeptide number and a 1.9-fold increase in total peak area of phosphopeptides in comparison with the results obtained under the conventional conditions. Since this approach is applicable to any single tip-based protocol without coupling with other enrichment methods, this simple strategy can be easily incorporated as an option into existing protocols for phosphopeptide enrichment, and would be suitable for high-throughput analysis in a parallel format.