2DE gels

There are a number of limitations that reduce the efficiency of 2 DE. Co-migration of proteins to the same location on a 2DE gel can occur. 

Figure 1 Schematic of the typical workflow of 2DE gel-based proteomics. This strategy includes flexible steps that can bypass 2DE, when desired, by directly proteolyzing cell lysates and cell fractions and analyzing by 2DLC-MS/MS.

Differentially expressed proteins are identified by comparison of 2DE gels from treated and control samples. By combining group-wise statistics with N-fold regulations, proteins are selected with a low p-value and are up or down regulated. Dose-responses or time-effects can be evaluated by special statistical methods. 

Figure 3.4 2DE-gels of SY5Y-TrkA cell lysate (A) or SY5Y-TrkB cell lysate (B). Separation of proteins was performed in the first dimension horizontal) by lEF and then in the second dimension (vertical) by SDS-PAGE. Representative pictures of whole cell lysates of... 

Construction of the 2DE gel image analysis packages 1985 Utilization of electrospray ionization (ESI) for mass spectrometry (MS) of large biomolecules... 

Figure 6. Peptide mass profiling of a silver stained protein spot from a narrow range, pH 4-7, 2DE separation of human heart (ventricle) proteins. A MALDI-TOF mass spectrum of tryptic peptides is shown, analyzed using a Micromass Tofspec 2E spectrometer (Manchester, UK) operated in the positive ion reflectron mode at 20 kV accelerating voltage with time-lag focusing enabled. The protein spot of interest was identified as human vimentin, (courtesy of J.A.Westbrook and R. Wait) (unpublished data). 2DE gels were silver stained using a modified Amersham Biosciences kit.

The expression patterns of glycoproteins can be monitored by 2DE in conjunction with MS. A general method for locating the glycoproteins in a mixture of protein on a 2DE gel is a specific glycan staining. Sensitive lectin (Corfield, 2000) and hapten recog-... 

Despite the development of new MS methods and instrumentation, the scanning and interpretation of proteins of 2DE gels has many limitations in the study of the proteome. Proteins of low abundance, high lipophilicity and low solubility (e.g. membrane proteins), and proteins that have an extremely low or high isoelectric point or have extremely low or high masses are not easily assessed with 2DE. 

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