2D-DIGE A Powerful Tool for Proteome Analysis

In the recent past, two dimensional gel electrophoresis has emerged as a powerful molecular biology tool for the comparative expression profiling of complex protein sample. It involves the separation as well as the resolution of diverse proteins sample on the basis of isoelectric points and molecular mass of protein in two dimension ways. In this way, it reflects the view of overall proteome status including differentiation in protein expression levels, post-translational modifications etc. Moreover, this allows the identification of novel biological signatures, which may give a particular identity of pathological background to cells or tissues associated with various types of cancers and neurological disorders. Therefore, by utilizing such tools, one can clearly investigate and compare the effects of particular drugs on cells of tissues and also one can analyze the effects of disease on the basis of variations in protein expression profile at broad spectrum. Recently, to get more error-less and accurate proteome profile, conventional 2-D gel electrophoresis has been enhanced with the inclusion of different types of protein labeling dyes which enables a more comparative analysis of diverse protein sample in a single 2-D gel. In this advanced technique (2-D-DIGE), protein samples are labeled with three different types of CyDyes (Cy2, Cy3, and Cy5) separately and combined and further resolved on the same gel. This will facilitate the more accurate spot matching on a single gel platform and will also minimize the experimental variations as commonly reported in the conventional 2D-gel electrophoresis. Therefore, in the present proteomic research era, 2D-DIGE has proved to be an extremely powerful tool with great sensitivity for up to 125 ng of proteins in clinical research volubility especially, neurological and cancer related disorders.

Author(s):  
Fatemeh Nasri ◽  
Maryam Zare ◽  
Mehrnoosh Doroudchi ◽  
Behrouz Gharesi-Fard

Background: Polycystic ovary syndrome (PCOS) is the most frequent endocrine disorder affecting 6–7% of premenopausal women. Recent studies revealed that the immune system especially CD4+ T helper cells are important in the context PCOS. Proteome analysis of CD4+ T lymphocytes can provide valuable information regarding the biology of these cells in the context of PCOS. Objective: To investigate immune dysregulation in CD4+ T lymphocytes at the protein level in the context of PCOS using two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). Methods: In the present study, we applied two-dimensional gel electrophoresis / mass spectrometry to identify proteins differentially expressed by peripheral blood CD4+ T cells in ten PCOS women compared with ten healthy women. Western blot technique was used to confirm the identified proteins. Results: Despite the overall proteome similarities, there were significant differences in the expression of seven spots between two groups (P <0.05). Three proteins, namely phosphatidylethanolamine-binding protein 1, proteasome activator complex subunit 1 and triosephosphate isomerase 1 were successfully identified by Mass technique and confirmed by western blot. All characterized proteins were over-expressed in CD4+ T cells from patients compared to CD4+ T cells from controls (P <0.05). In-silico analysis suggested that the over-expressed proteins interact with other proteins involved in cellular metabolism especially glycolysis and ferroptosis pathway. Conclusion: These findings suggest that metabolic adjustments in CD4+ T lymphocytes, which is in favor of increased glycolysis and Th2 differentiation are important in the context of PCOS.


PROTEOMICS ◽  
2006 ◽  
Vol 6 (5) ◽  
pp. 1631-1639 ◽  
Author(s):  
Anke Walz ◽  
Kai Stühler ◽  
Andreas Wattenberg ◽  
Eva Hawranke ◽  
Helmut E. Meyer ◽  
...  

2007 ◽  
Vol 154 (1) ◽  
pp. 6-21 ◽  
Author(s):  
Patricia Cuervo ◽  
Jose Batista de Jesus ◽  
Magno Junqueira ◽  
Leila Mendonça-Lima ◽  
Luis Javier González ◽  
...  

PROTEOMICS ◽  
2004 ◽  
Vol 4 (7) ◽  
pp. 1903-1908 ◽  
Author(s):  
Nazrul Islam ◽  
M. Lonsdale ◽  
N. M. Upadhyaya ◽  
T. J. Higgins ◽  
H. Hirano ◽  
...  

PROTEOMICS ◽  
2008 ◽  
Vol 8 (16) ◽  
pp. 3389-3396 ◽  
Author(s):  
Stephanie Weist ◽  
Murat Eravci ◽  
Oliver Broedel ◽  
Sandra Fuxius ◽  
Selda Eravci ◽  
...  

1996 ◽  
Vol 5 (1) ◽  
pp. 53-66 ◽  
Author(s):  
Johann Rudolf Frey ◽  
Klaus-Ulrich Hartmann ◽  
Ivan Lefkovits

Driven by our long-standing interest in identifying proteins of the immune system and in characterizing processes involved in lymphocyte differentiation, we studied protein expression in biosynthetically labeled fetal and newborn thymus by 2D gel electrophoresis. Autoradiographs of the gels were scanned with a densitometer and image analysis was performed using theKeplersystem. Calibrated polypeptide spot abundances (volumes) were compared to assesses qualitative and quantitative changes of the spot volumes. Among over 300 proteins evaluated at GD (gestation day) 13,15, and 17, there were sets of proteins that increased and others that decreased in intensity. We could in addition recognize proteins that were completely absent at GD 13 and/or 15 and that appeared thereafter to gradually increase in intensity. Conversely, various polypeptide spots present at early stages (at GD 13 and 15) disappear later (at GD 17 or at birth). Among the proteins that increase in intensity prevail molecules with masses less than 35 kD, whereas a considerable portion of those that decrease in intensity are characterized by masses above 60 kD. Spots reported in this communication were not defined beyond tagging them with numbers, which is a prerequisite to follow them up in the proteinpaedia developed in our laboratory. The next step will be to retrieve the coding sequences from the existing partitioned cDNA library (BW 5147) as well as from thymocyte subtraction libraries. We predict that among those polypeptides with varying intensity, important regulatory proteins in thymus development will be found.


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