Bioinertization of nanoLC/MS/MS systems by depleting metal ions from the mobile phases for phosphoproteomics

We have successfully bioinertized a nanoLC/MS/MS system for the highly sensitive analysis of phosphopeptides by depleting metal ions from the mobile phase. We found that not only the direct contact of phosphopeptides with metal materials, but also the indirect contact with HPLC pump through the mobile phase causes the significant loss in the recovery of phosphopeptides. Moreover, we observed that electrospray ionization was affected by the mobile phase containing multiple metal ions as well as the sample solutions contaminated with metal ions used by immobilized metal ion affinity chromatography for the phosphopeptide enrichment. To solve these problems, metal ions were depleted by inserting an on-line filter device containing metal chelating resins between the gradient mixer and the autosampler. As a result, we were able to increase the identification number of phosphopeptides by a factor of 1.5. This strategy would be applicable to highly sensitive analysis of other phosphorylated biomolecules by nanoLC/MS/MS.