2D DIGE vs LC-MS/MS
What are the differences between 2D DIGE and LC-MS/MS?
It is important to understand that 2D DIGE separates proteins by both MW and pI, thus offering very high resolutions of all proteins and their modified forms (such as PTMs and degradations) with a wide dynamic range. On the other hand, the mass spectrometry-based LC-MS/MS separates by peptides, which exceeds the number of proteins by several magnitudes.
It is obvious that the separation of proteins by both Mw and pI is of higher resolution than that of large number of small peptides by hydrophobicity only. Of course, both platforms will see better resolutions if coupled with removing abundant proteins. In addition, protein fractionation in combination with hydrophobicity can increase the peptide resolution in LCMCMS. However, all these extra steps will increase the cost and may compromise the reproducibility of data. The added benefits of 2D DIGE allow direct visualization of total protein profiling between the samples.
| 2D DIGE | LC-MS/MS | |
| Separate by | Proteins Molecular weight and pI | Peptides Hydrophobicity |
| Resolve high/low abundant proteins | Higher | Lower |
| Sensitivity for PTMs | Higher | Lower Needs to specify PTM |
| Detect protein degradation | Yes | No |
| Overall resolution | Highest for all sample types | Good for low to moderate complexity samples |
| Visualize protein profiling | Yes | No |
| Best fit for | Assess overall similarity between samples and identify changed proteins with high resolution | Identify all proteins in each sample and changed proteins between samples |