Week 5,6,7

Every week, since week 4 has been spent repeating the protein fractionation protocol for each sample, so as to separate out the proteins found in various areas of the cell (ie outer membrane, soluble proteins, inner membrane, and outer membrane washed proteins). Unfortunately, I only made it through the initial stages of protein fractionation on week 5 before I got sick. I went home on Wednesday afternoon and did not come back for the next week! It is now Wednesday of the 7th week and I am back in the lab at CMOP!

Yesterday, Tuesday July 23, was spent at the OHSU main campus in downtown Portland. Craig and I met up with two men working in the proteomics department of OHSU (Larry David and John Klimek) and they walked us through the process of MS-MS (MS=Mass Spectrophotometry) which would be used on the outer membrane wash portion of each of our samples that were successfully fractionated. In the case of our samples, this wash seemed to be the most active as far as Mn-oxidizing capability. It therefore made sense that we identify the proteins present in the active part of the samples in order to deduce which proteins may be responsible for oxidation of Mn.

Before placed into the MS-MS, each sample was prepared and trypsin-digested. Larry walked Craig and I through the preparation protocol, and allowed me to help prepare a few of the samples. Next the samples were to incubate overnight and that was the end of our work up on the hill! OHSU's lab now has all of our samples and will run them for us and send Craig the results in a few weeks time. Hopefully the identified gene sequences and proteins actually match previously existing databased proteins so that we can find out if our results mean anything. I guess we will found out soon. For now, its back to lab :)

By the way, for a full run through of how the MS-MS works, go to www.ohsu.edu/proteomics and click on the Mass_Spectrophotometry_Based_Proteomics.pdf found under the Proteomics Overview. This is full handout which describes in detail how the MS-MS works and what kind of information it yields.