CoolTip: Low-Temperature Solid-Phase Extraction Microcolumn for Capturing Hydrophilic Peptides and Phosphopeptides

Reversed-phase solid-phase extraction (SPE) techniques are commonly used for desalting samples before LC/MS/MS in shotgun proteomics. However, hydrophilic peptides are often lost during the desalting step under the standard SPE conditions. Here, we describe a simple protocol in which a stop-and-go extraction tip packed with a poly(styrene-divinylbenzene) copolymer disc is used at 4 °C during sample loading without any organic solvent. Using this method, which we designate as the CoolTip protocol, we identified 2.9-fold more tryptic peptides and 6.1-fold more tryptic phosphopeptides from HeLa lysates than the standard SPE protocol for hydrophilic peptides, with a mobile phase of less than 8% acetonitrile in LC/MS/MS. There was no decrease in the recovery of hydrophobic peptides. CoolTip also provided better quantitative reproducibility in LC/MS/MS analysis. We anticipate that this protocol will provide improved performance in many kinds of shotgun proteomics experiments. [Display omitted] •CoolTip, a StageTip with a poly(styrene-divinylbenzene) disc operated at 4 °C.•Identification of more 6.1-fold hydrophilic phosphopeptides from HeLa lysates.•No decrease in the recovery of hydrophobic peptides using the CoolTip protocol.•Better reproducibility in quantitative LC/MS/MS analysis. A desalting protocol to increase the recovery of hydrophilic peptides and phosphopeptides was developed by using SDB-StageTip at 4 °C. Using this protocol, 2.9-fold more tryptic peptides and 6.1-fold more tryptic phosphopeptides from HeLa lysates were identified for hydrophilic peptides.