Second dimension, proteomics

The beauty of 2D gel electrophoresis as a separation technique is the orthogonality of the two separation dimensions separation by charge (isoelectric point) in the first dimension, and separation by size in the second dimension. Two-dimensional gel electropheresis is the core separation technique for proteomics, along with HPLC (for preparative isolation). 

This chapter has presented several comprehensive 2DLC approaches combining a first-dimension IEX separation and a second-dimension RP separation for the analysis of complex protein mixtures typical in proteomics studies. Online ESI-TOF/MS detection provided sensitive detection and valuable qualitative information (MW) for proteins eluting from the MDLC system. Coordinated fraction collection and subsequent MS analysis of peptides produced by proteolysis of the fractions provided in-depth information on protein identification and a mechanism... 

Detection of the effluent in a 2D system is carried out at the end of the second dimension s column. UVand LIF are the most widely used and the simplest methods of detection for CE separations because they are performed on-column. MS detection, unlike UV and LIF, is carried out on the effluent as it exits the CE column. The direct coupling of CE with mass spectrometry has shown great potential in proteomic research (Janini et al., 2004). The method of choice for detection of peptides is MS-electrospray ionization (ESI). However, ESI requires a special interface between the CE column and the mass spectrometer that has proven not to be a simple matter (Issaq et al., 2004). 

Maximum reproducibility of 2-DE protein patterns in large-scale proteome analysis can be achieved by simultaneous electrophoresis of SDS-PAGE. Amersham Pharmacia Biotech (Piscataway, NJ) firm developed a multiple vertical second-dimension... 

Fig. 17.3. The infected bile-ome Two-dimensional electrophoresis gel of F. hepatica infected host bile. Run on a 17 cm, pH 3-10, IPG strip in the first dimension and then on 12.5% SDS-PAGE gel in the second dimension with Coomassie blue staining. Host and parasite proteins were identified via their peptide mass fingerprints (Morphew, 2004, unpublished). Valid parasite secreted proteins can only come from in vivo proteomics.

Fig. 6. Representative 2-DE gel of a rat liver proteome in the first dimension proteins were separated in a pH gradient (4-7), in the second dimension based on their molecular weight protein visualization was realized using fluorescent staining with ruthenium...

Figure 2. Two-dimensional BAC/SDS-PAGE of platelet membrane proteins. While not only large gels are recommended for complex samples, utilizing tube gels for the first dimension furthermore provides better resolution and more efficient transfer of proteins to the second dimension (less vertical smearing). (A) Small slab gel in the first dimension (7x7 cm). (B) Large tube gel in the first dimension (1 mm x 15 cm). (C) Summary of the human platelet membrane proteome study (Moebius et al. 2005). 158 proteins were exclusively identified from 2-DB gels, 65 from ID-PAGE. An overlap of 75 proteins was identified from both types of gels.

Although liquid chromatography techniques have become quite popular in the separation of peptides in complex protein digests, they are yet to make an impact for the separation of protein samples for proteome-wide applications. It is envisioned that in the future their application for protein separation will increase. Various combinations of reversed-phase (RP)-HPLC with ion-exchange, size-exclusion, chromato-focusing (CF), IEF, and capillary electrophoresis (CE) have emerged for 2D separation of complex mixtures of proteins and peptides. A recent addition in this field is the use of CF as the first dimension and RP-FIPLC as the second-dimension separation device.14 CF is a column-based liquid-phase separation technique, in which proteins are fractionated on the basis of differences in their p/values in a weak ion-exchange column. 

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