My previous post complained that our new mass spec data was difficult to interpret, partly because it gives no information about the relative abundances of the proteins it identifies in our Sxy prep. But it occurred to me that useful data about this may be available online.
This Sxy prep was purified from a standard E. coli K-12 strain growing probably in LB + an antibiotic. So I did some Google searching for "coli protein abundance" and easily found a year-old paper in Nature Biotechnology that compares the abundances of mRNAs and the corresponding proteins for E. coli and also for yeast (full text here). This paper nicely explains the reasons why mass spec alone can't really estimate protein abundance, and then describes a new method of combining data to does this. And a supplementary file provides all of their data on E. coli protein abundance ("APEX" estimates, as molecules per cell) in an Excel spreadsheet!
[Reading this paper also taught me that I was wrong to say in the previous post that peptide composition was calculated from each peptide's molecular weight. Instead each peptide is digested and put through a second mass spec that directly detects the amino acids it is composed of.]
What can we do with this information? We want to know, among other things, whether CRP is just one of many proteins that the purification procedure used for our Sxy prep fails to completely wash away, or whether our Sxy prep contains CRP because CRP specifically interacts with Sxy and thus co-purifies with it. If the latter, we expect our prep to contain more CRP than would be predicted based on its usual abundance in cells. Thus I think we should check the APEX abundances of all the proteins identified in our sample. If CRP has a much lower APEX value than the other contaminating proteins the mass spec analysis identified, we can suspect that CRP does interact with Sxy.
Of course, lots of confounding factors are likely to affect the efficiency with which different proteins will be removed by the purification procedure. Unfortunately our 'informed' guesses about these factors are not informed by much. But I think this is still worth a try.
Why I'm Marching for Science
16 hours ago in Angry by Choice