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CoolTip: Low-Temperature Solid-Phase Microextraction Tip for Capturing Hydrophilic Phosphopeptides: 2

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: PXD028871 | 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]

PMID: 34740827

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Project description:Proper sample preparation protocols represent a critical step for LC-MS-based proteomic study designs and influence the speed, performance and automation of high-throughput data acquisition. The main objective of this study was to compare two commercial solid phase extraction (SPE)-based sample preparation protocols (comprising SOLAµTM HRP SPE spin plates from Thermo Fisher Scientific and ZIPTIP® C18 pipette tips from Merck Millipore) for analytical performance, reproducibility and analysis speed. The house swine represents a promising animal model for studying human eye diseases including glaucoma and provides excellent requirements for the qualitative and quantitative MS-based comparison in terms of ocular proteomics. In total 6 technical replicates of two protein fractions [extracted with 0.1% dodecyl-ß-maltoside (DDM) or 1% trifluoroacetic acid (TFA)] of porcine retinal tissues were subjected to in-gel trypsin digestion and purified with both SPE-based workflows (N=3) prior to LC-MS analysis. On average, 550±70 proteins (1512±199 peptides) and 305±48 proteins (806±144 peptides) were identified from DDM and TFA protein fractions, respectively, after ZIPTIP® purification, and SOLAµTM workflow resulted in the detection of 513±55 proteins (1347±180 peptides) and 300±33 proteins (722±87 peptides), respectively (FDR<1%). Venn diagram analysis revealed an average overlap of 65±2% (DDM fraction) and 69±4% (TFA fraction) in protein identifications between both SPE-based methods. Quantitative analysis of 25 glaucoma-related protein markers also showed no significant differences (P>0.05) regarding protein recovery between both SPE methods. However, only glaucoma-associated marker MECP2 showed a significant (P=0.02) higher abundance in ZIPTIP®-purified replicates in comparison to SOLAµTM-treated study samples. Nevertheless, this result was not confirmed in the verification experiment using in-gel trypsin digestion of recombinant MECP2 (P=0.24). In conclusion, both SPE-based purification methods worked equally well in terms of analytical performance and reproducibility, whereas the analysis speed and the semi automation of the SOLAµTM spin plates workflow is much more convenient in comparison to the ZIPTIP® C18 method.

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