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What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

ABSTRACT: Two-dimensional gel electrophoresis was instrumental in the birth of proteomics in the late 1980s. However, it is now often considered as an outdated technique for proteomics-a thing of the past. Although this opinion may be true for some biological questions, e.g., when analysis depth is of critical importance, for many others, two-dimensional gel electrophoresis-based proteomics still has a lot to offer. This is because of its robustness, its ability to separate proteoforms, and its easy interface with many powerful biochemistry techniques (including western blotting). This paper reviews where and why two-dimensional gel electrophoresis-based proteomics can still be profitably used. It emerges that, rather than being a thing of the past, two-dimensional gel electrophoresis-based proteomics is still highly valuable for many studies. Thus, its use cannot be dismissed on simple fashion arguments and, as usual, in science, the tree is to be judged by the fruit.

SUBMITTER: Marcus K

PROVIDER: S-EPMC7563651 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

Marcus Katrin K Lelong Cécile C Rabilloud Thierry T

Proteomes 20200806 3

Two-dimensional gel electrophoresis was instrumental in the birth of proteomics in the late 1980s. However, it is now often considered as an outdated technique for proteomics-a thing of the past. Although this opinion may be true for some biological questions, e.g., when analysis depth is of critical importance, for many others, two-dimensional gel electrophoresis-based proteomics still has a lot to offer. This is because of its robustness, its ability to separate proteoforms, and its easy inter ...[more]

PMID: 32781532

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Project description:BackgroundThe low concentration and highly hydrophobic nature of proteins in lipid raft samples present significant challenges for the sensitive and accurate proteomic analyses of lipid raft proteins. Elimination of highly enriched lipids and interfering substances from raft samples is generally required before mass spectrometric analyses can be performed, but these procedures often lead to excessive protein loss and increased sample variability. For accurate analyses of the raft proteome, simplified protocols are needed to avoid excessive sample handling and purification steps.ResultsWe have devised a simple protocol using a 'tube-gel' protein digestion that, when combined with mass spectrometry, can be used to obtain comprehensive and reproducible identification and quantitation of the lipid raft proteome prepared from neonatal mouse brain. Lipid rafts (detergent-resistant membranes using Triton X-100 extraction) prepared from neonatal mouse brain were directly incorporated into a polyacrylamide tube-gel matrix without prior protein separation. After in-gel digestion of proteins, nanospray LC-MS/MS was used to analyze the extracted peptides, and the resulting spectra were searched to identify the proteins present in the sample. Using the standard 'label-free' proteomics approach, the total number of MS/MS spectra for the identified proteins was used to provide a measure of relative protein abundances. This approach was successfully applied to lipid rafts prepared from neonatal mouse brain. A total of 216 proteins were identified: 127 proteins (58.8%) were predicted to be membrane proteins, or membrane-associated proteins and 175 proteins (~80%) showed less than a 2-fold variation in the relative abundance in replicate samples.ConclusionThe tube-gel protein digestion protocol coupled with nanospray LC-MS/MS (TubeGeLC-MS/MS) offers a simple and reproducible method for identifying and quantifying the changes of relative abundances in lipid raft proteins from neonatal mouse brain and could become a useful approach for studying lipid raft proteins from various tissues.

Dataset Information

What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

Publications

What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

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