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Quantitative Proteomic Analysis of Membrane Proteins Based on Gel and Shotgun Techniques

Trong Khoa Pham and Phillip C. Wright. Chemical and Process Engineering, The University of Sheffield, CHELSI, The Sheffield Bioincubator, 40 LeavyGreave Road, Sheffield, S3 7RD, United Kingdom

Most membrane proteins play essential cellular functions. However, from a proteomics aspect, the analysis of membrane proteins is still challenging due to the hydrophobic nature of membrane proteins that makes it difficult for proteomic analysis, especially for quantitative proteomics. Until now, proteomic analysis of membrane proteins has mostly been based on 2-DE followed by mass spectrometry. However, this technique is limited by the difficulty of solubilizing and resolving membrane proteins on gels, furthermore, identification of proteins based on gels is often tedious. Unlike 2-DE techniques, shotgun proteomics offers a useful alternative to gels. Although, recently, many different shotgun methods have been used to analyze enriched membrane fractions using detergents, organic solvents, and additives, in the present of trypsin or other proteolytic agents, none of these methods has been used for quantitative membrane proteomic analysis yet.

Sulfolobus solfataricus is a member of archaeal domain, that has been received much attention since the mechanisms relating to information processes represent a simpler version of eukaryotic equivalents. S. solfataricus is a thermoacidophilic crenarchaeon that grows optimally at conditions of 80oC and pH 3. To survive under these extreme conditions at high temperature and low pH, S. solfataricus needs to optimise its biological behaviour, especially at the bioenergetic aspect. How it manages to do this is not yet clear. Additionally, most bioenergetic processes, especially oxidative processes, are performed via membrane proteins, but the numbers of these proteins that have been experimentally identified as well as quantified are still low. Therefore, development of techniques to overcome this issue is necessary.

We describe a method for extracting S. solfataricus membrane proteins, as well as applying both techniques based on 2-DE and shotgun workflows for quantitative proteomic of S. solfataricus membranes. Here, for the first time, the application of isobaric tags for relative and absolute quantitation (iTRAQ) for quantitative analysis of membrane proteins is performed. A comparison between 2-DE and iTRAQ techniques for quantitative proteomics of membranes is also discussed. Moreover, the application of a new MS instrumentation based of an ion trap with electron transfer dissociation (ETD) is also investigated in comparison with Qq-TOF instruments employing collision induced dissociation (CID).