Functional Proteomics

Questions: 1. Describe a proteomics experiment that you think would be interesting to perform in our weeklong practical session?2.Suggest a follow-up experiment you could do to confirm your results?3.Why should you buy a mass spectrometer for your proteomics laboratory?4.What important things you learned from your practical class?5.Explain how you would design an experiment to decide whether the new enzyme is a Histidine-specific protease? Answers: 1. The well-established method to investigate proteomics is mass spectrometry. There are also several strategies present which can conduct proteomic research. A few years ago proteomics was mainly a qualitative discipline. The proteomic experiment characteristically involved identification of proteins as many as possible in a particular protein complex, tissue lysate, organelle and cell. While obtaining the protein identities from any type of protein mixture an enzymatic digestion step is normally involved which ultimately yields a large collection of proteolytic peptides that are analyzed by shotgun proteomics. This technique can be incorporated in the proteomics experiment and it would be interesting to perform. 2. After performing 2-DE and mass spectrometry to examine differential protein expression between the two strains of Giardia with different types of virulence phenotypes, a Quantitative PCR can be performed as a follow up experiment to confirm the results. In Quantitative PCR total RNA was isolated from the batch culture of the Giardia sp. Then reverse transcription (RT) was carried out with a reaction mixture which contain 5g total RNA, 0.25mM deoxynucleoside triphosphates (dNTPs), 50 nM of RT primer, 0.75 U/l reverse transcriptase enzyme, 0.05 M DTT and 0.2 U/l RNase Out. After this an incubation of the RT reaction mixture at 50C was done for 30 min, and then the reaction stopped at 70C for 15 min. 3. If I had funding to buy mass spectrometer while establishing a proteomics laboratory I will buy a mass spectrometer because mass spectrometry is an exceedingly specific and sensitive analytical technique that can be used to determine precisely the quantities and identities of the compounds that are present in the sample. In comparison to many traditional technologies mass spectrometry delivers analytical more sensitive result and is very rapid and fast less time taking process. This is the reason that mass spectrometry has a variety of application in pharmaceutical and clinical laboratories. 4. The most important thing that I have learned in my practical class is in spite of a well conserved and core sequence of the genes there still remains very significant variation in the genome of the Giardia isolates. These variations can be observed in gene content, structural chromosomal variations, gene polymorphisms, and repertoires of the surface molecule. From this practical class the annotation of the Giardia genome has become clear and improved in according to me and this practical session also enabled the identification of the functionally important variation of the Giardia genome. 5. Histidine-specific protease is an enzyme which cleaves the peptide bonds present in the proteins in which the histidine serves as the nucleophilic amino acid at the active cite of the protein. So to find out whether it is a worth to buy the Histidine-specific protease or not we can use it against a protein which contain the histidine amino acid. Then the enzyme will cleave the peptide bond as a result of that the N- terminus of the protein bond will react with the hydrogen of the water molecules and the C-terminus will become free which will have a huge impact over different physiological process like blood coagulation, immune response and digestion.