CoolTip: Low-Temperature Solid-Phase Microextraction Tip for Capturing Hydrophilic Phosphopeptides

We developed a simple temperature controlled desalting strategy to cover from hydrophilic to hydrophobic peptides. Reversed-phase solid-phase extraction (SPE) techniques are commonly used for shotgun-proteomics using liquid chromatography/tandem mass spectrometry. In reversed-phase chromatography, solid-phase temperature is one of the parameters that influences peptide retention, and the analytical column temperatures have been well optimized for the peptide separations. However, little attention has been given to the temperatures of solid-phase extraction columns for sample preparations. We showed that the recovery of hydrophilic (phospho)peptides improved with lower temperature SPE-tips (CoolTips) and identification coverage of the hydrophilic (phospho)peptides can be extended without sacrificing the recovery of hydrophobic peptides. CoolTip also provided better quantitative reproducibility of the peptides, and we envision this protocol can improve the performance of any kinds of proteomics experiments.