Proteomics

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

ABSTRACT: 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.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama 

PROVIDER: PXD027112 | JPOST Repository | Thu Oct 28 00:00:00 BST 2021

REPOSITORIES: jPOST

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

Ogata Kosuke K   Ishihama Yasushi Y  

Molecular & cellular proteomics : MCP 20211103

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  ...[more]

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