Today I consulted two very helpful experts in mass spectrometric to find out the sequences of al;l the peptides in the mix (more or less) methods. I wanted to find out if we really have a hope of identifying DNA uptake proteins by crosslinking them to DNA. It looks promising.
The plan is to incubate competent cells with DNA, and then add formaldehyde, which will create crosslinks between DNA and the proteins it's in contact with. We'll then dissolve the cells and pull out the DNA with its attached proteins. Then we'll get rid of the DNA and undo the crosslinks (by boiling the mix), leaving us with a little tube containing a mixture of DNA-contacting proteins of unknown identities. Then we'll digest the proteins with the protease trypsin, which will cut them into predictable pieces.
We'll use a combination of HPLC (high performance liquid chomatography) and mass spectrometry to find out the amino acid sequences of all the peptides in the mix. By comparing these with the known sequences of all the proteins specified by the genome, we'll know what proteins the peptides came from. By comparing results with DNAs that either do or don't have a USS (or have a variant USS) and with cells carrying different mutations, we can infer a lot about the specific interactions (I hope).
We don't need to invest in equipment for or learn how to do the HPLC and mass spec; we can pay local experts to do it for us.
One issue we'll need to grapple with is the small amounts of protein our 'fishing' technique is likely to produce. I think we can scale up, but that's always a source of problems. Another issue is stopping the cells from quickly sucking the DNA all the way inside - we may be able to control this by initially using cells with uptake mutations, or by sticking the DNA onto beads that are too big for the cells to take up.
Friday Fabulous Flower - Redbuds
1 day ago in The Phytophactor