Extending the identification coverage of hydrophilic proteome and phosphoproteome via a graphite solid phase extraction StageTip

Desalting and concentrating peptides using reverse phase chromatography prior LC-MS/MS analysis is a critical step to improve the sensitivity of peptide detection. However, hydrophilic peptides are not easy to retain on the conventional solid phase extraction (SPE) StageTip using C18 as materials during the desalting step. In this study, graphite carbon was utilized as a SPE StageTip with strong hydrophobic adsorbent, which doubled the identification number of multiply phosphopeptides compared to the number using C18 SPE StageTip. In addition, (phospho)peptides with lower GRAVY value and shorter peptides length were enriched using the graphite SPE StageTip. Finally, a total of 7,617 phosphopeptides were identified, which covers both hydrophilic and hydrophobic properties. The complementary affinity of different desalting materials including C18, SDB-XC, and graphite was further demonstrated to deepen the coverage of proteome and phosphoproteome.