I keep asking myself "Why aren't I getting any science done?". The answer seems to be partly that I've been bogged down with reviewing manuscripts and reading theses, partly that preparing for next term's teaching is starting to loom large, and partly that I've been doing incremental bits of work on various fronts that don't feel as much like research as I would like.
Today I struggled to understand what we could and couldn't learn from the mass-spec analysis of a purified protein. This is a His-tagged E. coli Sxy protein, purified from E. coli cells using a nickel-affinity column. The prep has two odd features. First, in a gel it gives two bands, both close to the size of the expected Sxy-His product and both of roughly equal intensity. We want to find out what distinguishes these two proteins. Second, the prep behaves in band-shift assays as if it also contains a small amount of another protein, CRP. There's not enough CRP to detect in a gel (I forget whether we can detect it with an antibody in Western blots). We hoped the mass spec would tell us whether the prep does indeed contain enough CRP to explain the bandshift results.
Now that I have a better idea what the mass spec analysis does and doesn't do, I see that it can't give us very useful answers.
Here's what it does: All the protein in the prep is first digested into peptides by the protease trypsin. Mass spec analysis of this mixture then determines the exact (to about 7 significant figures) molecular weight of each detectable peptide. The threshold of detection is very low; I think the post-doc who best understands this told me that it's about a femtomole of peptide. Software then calculates the amino acid composition or compositions that could give this molecular weight. (Because different combinations of amino acids can sum to the same weight, several alternative compositions may be possible.)
Other software has analyzed the E. coli proteome, calculating the composition of every peptide that could be produced by trypsin digestion. This database is then compared with the observed peptides in the mass spec sample, to see which mass spec peptides could have come from which E. coli proteins. If a significant number of mass spec peptides match a particular E. coli protein, the software reports that that protein was likely present in the sample.
We have two main problems with the results from our sample. The first is because the mass spec analysis is so sensitive - it identified about 150 proteins in our sample! The second is because the report gives no indication of the relative amounts of the different peptides in the sample - we have no idea which proteins are abundant and which are present only in femtomole amounts. Sxy is one of the 150, which reassures us that we have purified the right protein. So is CRP. Finding that CRP was absent from the prep would have been very significant, because it would have meant that CRP could not be responsible for the bandshifts the prep causes, but finding that it is present doesn't advance things very much. This is largely because we get no information about how much CRP is present, relative to Sxy and to all the other proteins.
We also have some practical problems in interpreting the data. First, the results file is full of hyperlinks, but none of them work (we're 'not authorized'), so we can't tell what we would learn by clicking on them. Second, some of the peptides seem to not match the indicated protein - we don't know if there's a flaw in the software of if we're just misinterpreting the data. So more consultation with the person who does the mass spec analysis is needed.
We had been planning to cut out each of the Sxy-sized bands from a gel and run them separately through the mass spec analysis. But if each of these excised bands is even slightly contaminated with protein from the other, the mass spec will detect them both in both preps. Excising the bands will remove (or at least greatly decrease) most of the contaminating proteins, so he results should be much simpler, but I don't know how much we can learn about the identities of the proteins in these bands, especially if one or both of them differs in sequence from the predicted E. coli proteins.
Luckily the post-docs have lots of ideas for tests that don't rely on mass spec.
Friday fabulous flower - yellow lawn edition
5 hours ago in The Phytophactor