**6. References**

352 Gel Electrophoresis – Advanced Techniques

endoglycosidase PNGase to cleave N-linked oligosaccharides from the protein backbone and the second is based on enzymatic removal of sialic acid with neuraminidase. As shown in figure 14A, SAA4 is usually expressed in HDL as 6 isoforms, three with molecular masses about 18k and three with molecular masses about 11k. After PNGase treatment it was clearly shown that the 18k isoforms are depending on N-linked glycosylation (Fig 14A). Another glycosylated protein in HDL is apo C-III that can be found as three isoforms; one di-sialylated, one mono-sialylated and one minor non-sialylated form (Bruneel et al., 2008). This was demonstrated by treatment with neuraminidase, which induced a mobility shift with the loss of the two sialylated isoforms and a substantial increase of the non-sialyated

Fig. 14. 2-DE mass and charge mobility shift assays to demonstrate glycosylated protein

A: SAA4 analyzed by 2-DE and silver stained. N-linked glycosylated serum amyloid A4 (SAA4) isoforms shown by deglycosylation with PNGase. B: Apo C-III analyzed by 2-DE

Overall the binding of plasma proteins to nanoparticles, based on our findings, seems to vary with origin, surface properties and size of the particles. A large portion of the interacting proteins we identified by 2-DE/MS are proteins involved in the immune defense and reverse cholesterol transport to the liver, but we also identified proteins mediating brain uptake. Most likely these protein patterns of the nanoparticles represent a mixture of particle-protein and protein-protein interactions. Extensive research in this field is therefore needed before conclusions could be drawn regarding potential health effects of nanoparticles and their associated protein "corona". One major difficulty to overcome is how to characterize the particles used in different studies in such a way that comparisons

and Western blots. Sialylated apo C-III isoforms shown by desialylation with

apo C-III form (figure 14B).

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neuraminidase.

**5. Conclusions and future research** 


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**19** 

*India* 

**Two Dimensional Gel** 

**Electrophoresis in Cancer Proteomics** 

Shenbagamoorthy Sundarraj1 and Ramasamy Thirumurugan2

*2Department of Animal Science, Bharathidasan University, Tiruchirappalli* 

Two-dimensional electrophoresis (2-DE) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. This technique sort's protein according to two independent properties in two discrete steps: the first-dimension step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI); the second-dimension step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to their molecular weights (Mr, relative molecular weight). Each spot on the resulting two-dimensional array corresponds to a single protein species in the sample. Thousands of proteins can thus be separated, and information such as the protein pI, the apparent molecular weight, and the amount of each protein obtained. The separation of proteins by 2-DE dates back to the 1950s. The rst 2-DE technique was developed by Smithies and Poulik in 1956 and O'Farrell, 1975 and Klose, 1975 significantly modified this method to elucidate protein profile. In the original technique, the first-dimension separation was performed in carrier ampholyte-containing polyacrylamide

The power of 2-DE as a biochemical separation technique has been recognized virtually since its introduction. Its application, however, has become significant only in the last few years because of a number of developments. The introduction of immobilized pH gradients and Immobiline™ reagents brought superior resolution and reproducibility to firstdimension IEF. Based on this concept, Görg *et al*., 1989 and Gorg, 1991 developed the currently employed 2-D technique, where carrier ampholyte-generated pH gradients have been replaced with immobilized pH gradients and tube gels replaced with gels supported by a plastic backing. New mass spectrometry techniques have been developed that allow rapid identification and characterization of very small quantities of peptides and proteins extracted from single 2-D spots. More powerful, less expensive computers and software are now available, rendering thorough computerized evaluations of the highly complex 2-D patterns economically feasible. Data about entire genomes (or substantial fractions thereof) for a number of organisms are now available, allowing rapid identification of the gene encoding a protein separated by 2-DE. The World Wide Web provides simple, direct access

**1. Introduction** 

gels cast in narrow tubes.

Soundarapandian Kannan1, Mohanan V. Sujitha1,

*Department of Zoology, Bharathiar University, Coimbatore* 

*1Proteomics and Molecular Cell Physiology Lab* 

Zensi, A.; Begley, D.; Pontikis, C.; Legros, C.; Mihoreanu, L.; Buchel, C. & Kreuter, J. (2010). Human Serum Albumin Nanoparticles Modified with Apolipoprotein a-I Cross the Blood-Brain Barrier and Enter the Rodent Brain. *Journal of Drug Targeting*, Vol.18, No.10, pp. 842–848, ISSN 1029-2330
