Two Dimensional ElectrophoresisEdit
Two Dimensional Electrophoresis is a time-tested laboratory method for separating complex mixtures of proteins by two orthogonal properties: isoelectric point and molecular weight. Developed in the late 20th century, the technique became a workhorse of proteomics, allowing researchers to visualize hundreds to thousands of protein species in a single digestible map. By pairing a high-resolution separation by pI in the first dimension with separation by size in the second, scientists can compare patterns across samples and identify proteins that change in expression, modification, or processing.
In practice, two-dimensional electrophoresis serves as a bridge between the crude complexity of a cell’s proteome and the precise identifications that modern mass spectrometry can deliver. After separation, protein spots are visualized by staining, quantified, and often excised for identification by methods such as mass spectrometry or MALDI-TOF. The technique has evolved with refinements such as differential labeling to improve quantitative comparisons and with ongoing efforts to extend its reach to challenging classes of proteins.
Core principles
Two-Dimensional Gel Electrophoresis (2-DE) combines two distinct, sequential separation steps that are orthogonal to each other:
First dimension: isoelectric focusing on immobilized pH gradient strips. Proteins migrate in a pH gradient until they reach their isoelectric point (pI), the pH at which their net charge is zero. This step resolves proteins primarily by charge properties, yielding a horizontal separation on the resulting gel strip. See Isoelectric focusing for a broader discussion of the technique.
Second dimension: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After equilibration, the strip is laid on top of a conventional polyacrylamide gel, and proteins are separated vertically by molecular weight under denaturing conditions. This orthogonal step complements the first by resolving proteins based on size.
The net effect is a two-dimensional array of spots, each representing a protein species or a closely related group of isoforms. Spot shapes and intensities reflect abundance and post-translational modification states, making 2-DE a powerful survey tool for differential expression.
Methodology and workflow
Sample preparation and solubilization: Proteins are extracted under conditions that preserve their native modifications as much as possible while ensuring solubility in the gel matrix. Membrane proteins, hydrophobic species, or very high- or low-molecular-weight proteins pose particular challenges and may require specialized buffers.
First-dimension separation: IPG strips (immobilized pH gradient) are rehydrated with the protein sample and subjected to an electric field. The pH gradient concentrates proteins at their pI, producing a pH-based separation.
Second-dimension separation: The focused strip is equilibrated in buffers that impart uniform negative charge (via SDS) and then placed on a standard SDS-PAGE gel. Proteins separate according to molecular weight.
Visualization and analysis: Gels are stained using compatible dyes (e.g., Coomassie, silver, or fluorescent stains). Digital imaging enables spot detection and densitometry. Software tools align and compare spots across gels to identify differential expression patterns. See proteomics for how this fits into broader workflows.
Spot excision and identification: Spots of interest are excised and digested (commonly with trypsin). The resulting peptides are analyzed by mass spectrometry to determine protein identity, often employing MALDI-TOF or LC-MS/MS pipelines for peptide mass or sequence information.
Variants and enhancements: Differential in-gel electrophoresis (DIGE) uses covalent fluorescent labeling to visualize multiple samples on the same gel, improving quantitative accuracy by reducing gel-to-gel variability. See DIGE for more.
Variants and limitations
2D-DIGE and other labeling approaches: By labeling different samples with distinct fluorescent dyes, researchers can run several samples side-by-side on a single gel, enabling more precise quantitative comparisons. This approach reduces the technical variability from gel-to-gel differences.
Limitations and challenges: The technique has a relatively low throughput compared with modern LC-MS/MS-based proteomics. It can be labor-intensive and time-consuming, and it tends to underrepresent highly hydrophobic, very large, or very small proteins and proteins with extreme pI values. Spot co-migration can blur interpretations, as multiple proteins may occupy a single spot. Controversies in the field often center on whether 2-DE remains the most efficient route for comprehensive proteome coverage or whether shotgun approaches provide superior depth for many applications.
Reproducibility and data interpretation: Gel-to-gel variability historically complicates quantitative comparisons. Modern approaches mitigate this with internal standards (as in DIGE) and advanced image analysis, but some observers remain skeptical about the dynamic range and accuracy of spot-based quantitation for certain biological questions. See reproducibility for related discussions.
Membrane and post-translational modifications: Hydrophobic membrane proteins and certain post-translationally modified species can be underrepresented or show smeared spots, requiring alternative or complementary methods to obtain a complete picture of the proteome. See post-translational modification for context.
Applications in research and industry
Differential expression studies: By comparing proteomes from different conditions (for example, healthy versus diseased tissues), researchers can identify proteins that respond to an intervention or pathophysiology.
Protein modification mapping: Changes in charge or mass due to phosphorylation, glycosylation, or other modifications can shift spot position or mass in the second dimension, enabling targeted follow-up analyses.
Biomarker discovery: Earlier proteomics efforts used 2-DE to identify candidate biomarkers that were later validated by more expansive, high-throughput methods. The technique helped build foundational datasets that informed modern biomarker pipelines.
Industrial and academic contexts: 2-DE remains a reliable, well-understood method in many labs worldwide, appropriate for exploratory workflows, method development, and training in proteomics concepts.