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Native detection of protein O-GlcNAcylation by gel electrophoresis.

ABSTRACT: O-GlcNAcylation is an abundant and dynamic protein posttranslational modification (PTM), with crucial roles in metazoans. Studies of this modification are hampered by the lack of convenient methods for detecting native O-GlcNAcylation. Here, we describe a novel gel-based approach, Separation of O-GlcNAcylated Proteins by Polyacrylamide Gel Electrophoresis (SOPAGE), which enables detection of O-GlcNAc levels and dynamics.

SUBMITTER: Fu C

PROVIDER: S-EPMC7611126 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Native detection of protein <i>O</i>-GlcNAcylation by gel electrophoresis.

Fu Chuan C van Aalten Daan M F DMF

The Analyst 20201001 21

O-GlcNAcylation is an abundant and dynamic protein posttranslational modification (PTM), with crucial roles in metazoans. Studies of this modification are hampered by the lack of convenient methods for detecting native O-GlcNAcylation. Here, we describe a novel gel-based approach, Separation of O-GlcNAcylated Proteins by Polyacrylamide Gel Electrophoresis (SOPAGE), which enables detection of O-GlcNAc levels and dynamics. ...[more]

PMID: 33103664

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Project description:BackgroundProteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE).ResultsSeparation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.ConclusionThe present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

Dataset Information

Native detection of protein O-GlcNAcylation by gel electrophoresis.

Publications

Native detection of protein <i>O</i>-GlcNAcylation by gel electrophoresis.

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