**5. Conclusion**

622 Biomedical Science, Engineering and Technology

amino acid X can have when forming part of the G–X–G tripeptide in extended

<sup>20</sup> ,max 1 ,max *i i*

⎛ ⎞ <sup>⋅</sup> = ⋅ ⎜ ⎟ <sup>⋅</sup> ⎝ ⎠

*n s*

*n s*

<sup>∑</sup> <sup>∑</sup> (16)

*aai i j j j A*

= ∈

After that, a cluster analysis was performed in order to classify them as low, medium or high hydrophobicity proteins. This analysis showed that the highly abundant proteins, i.e. albumin, immunoglobulins, fibrinogen and haptoglobin, exhibited a medium hydrophobicity, and thus they fell in the same cluster. With this information, a HIC step was designed to deplete highly abundant proteins from rat plasma samples. The HIC step consisted of stepwise elution to separate the three groups of proteins (low, medium and high hydrophobicity) using a maximum concentration of 2 M ammonium sulfate, and concentration for elution of 0.6 M (to desorb low hydrophobicity proteins), 0.5 M (to desorb medium hydrophobicity proteins), and 0.0 M (to desorb the highly hydrophobic

Finally, the depleted samples were analyzed by 2DGE and the performance of the HIC pre-fractionation step was compared with that exhibited by a commercial immunoaffinity column. The reproducibility of 2DGE was similar to that obtained from immunoaffinity depleted plasma. However, HIC was more successful in depleting albumin and α-1-antitrypsin. Besides, HIC resulted in a much lower increment of immunoglobulin and haptoglobin abundances than the immuno-affinity column. Then, HIC depletion allowed detecting twice the number of protein spots than immuno-affinity depletion did. Therefore, HIC could be used as a depletion method complementary to affinity columns. The operating conditions in HIC could be optimized in order to maintain the high number of spots that are detected if HIC is used as the sole depletion method. Finally, given the relatively low cost of HIC supports and HIC operation, its use could be proposed as a convenient choice for depleting highly abundant proteins in plasma samples prior to

Protein–protein interactions are essential in biological processes. All the interactions in a cellular system are known as protein interaction network or 'interactome'. In Biomedicine there is great interest in recognizing these interactions, aiming to establish the role they play in certain diseases. The traditional approaches to study protein-protein interactions are the antibody pull-down method (APD) and the yeast two-hybrid method (YTH). Despite their popularity, these methods have some disadvantages. It is very likely that a protein forms part of different complexes; then, in an APD experiment, antibodies targeting such a protein will pull down together all the complexes where the protein participates, making them appear to be part of a single large complex, confusing the biological interpretation of the results. The YTH is an "*in vivo*" method that allows detecting only binary interactions. It tends to give false positives and is limited to binary interactions. Therefore it is not useful in studying the

dynamics of complex formation triggered by different stimuli (Corvey et al., 2005).

Liu et al. (2008) investigated the potential of chromatography to allow the simultaneous examination of multiple protein complexes along with comparing and validating results from the traditional methods. Since protein complexes remain intact during mild forms of

φ

*ASH*

conformation (Miller et al., 1987).

proteins).

2DGE-based proteomics.

**4.2.2.2 Analysis of protein interaction networks by HIC** 

HIC is a powerful tool for purifying macromolecules of biomedical interest whose potential has been relatively under-exploited so far. Its applications are diverse, including industrial processes as well as analytical methods. The performance of HIC can be improved by optimizing the supports and the operation mode considering the hydrophobicity of the macromolecule to be separated. Research on optimization of HIC for biomedical applications should be encouraged, since this method allows reducing production cost of biopharmaceuticals such as antibodies and therapeutic proteins.
