Medium/Heavy comparison with XPress

[Damon May]

I've got triple-labeled data, heavy-medium-light K. We've discussed before the possibility of supporting this type of data fully, and I know that's not possible yet.

However, I would like to compare medium with heavy (specifically, I'd like to assess M/(M+H)). This is only a comparison of two quantities, but I'm having a hard time figuring out how I might do it with XPress.

I tried running XPress twice (H/L and M/L) and using the values for H from one run and M from the other. That did not behave well at all, and when I sanity checked by comparing H/L ratios derived from one XPress run with H/L ratios derived from the H from an H/L run and the L from a M/L run, the low correlation (~0.3) made me realize that I should not expect this to work well.

I considered re-running Comet, pretending that there's no light label, and running XPress on the results. This would certainly "work", but I would lose quantitation from all PSMs in which only the light was identified; that's likely quite a lot of peptides.

My only other idea is to mutilate the PepXML from my current (L-M-H) Comet searches, dummying them up so that it looks like I ran an M-H only search, and creating dummy M or H IDs for everything that's currently an L ID. That's a really ugly solution, though, and I'm worried it'll fail for some reason I haven't thought of.

Can anybody think of another way to do this?

Thanks,
Damon

[Amit Yadav]

Hi Damon,

Have you tried checking the correlation for only those ratios where all three labels (L M H) are present?. The reason could be that finding any two pairs may have higher chances of false positives which may be distorting your real ratios and therefore resulting in low correlation.

Since you wanted H/M, why do you expect quantitation to suffer when you ignore light label?

I don't have idea about this mutilation trick.

If possible, a triple SILAC search can be done with ProteinPilot and any ratios can be calculated even post search (M/L, H/L, H/M or any other combination)

In freely available software solutions, MaxQuant can be used.

[Damon May]

Thanks for the response, Amit. Sorry, I'm not sure what you mean by "correlation for only those ratios where all three labels (L M H) are present". By definition, I'm only comparing ratios that were present as H/L in one XPress run and M/L in the other (and so L M H are present in one run or the other). Do you mean ratios where all 3 labels have PSMs? That seems a bit stringent.

It's not that I expect the quantitation to suffer when I ignore the light label. It's that, demonstrably, when I compare the heavy from a H/L XPress run and the light from an M/L run, I get very different results from the H/L run's ratio. Meaning that, presumably, the L quantity that's measured is varying significantly across the two runs. For that reason, I expect the H from the H/L run also to be difficult to compare with the M from the M/L run.

MaxQuant might be the better option for me. Thanks for the suggestion!

Damon

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[Damon May]

Just to clarify and add some detail: if I run XPress twice on the same (very large, multi-sample) pepXML file, once with proper arguments for H/L (8Da separation) and once with proper arguments for M/L(8Da separation), and then I compare the logs of the light areas determined with the two methods, PSM by PSM, I get a correlation coefficient of 0.87. The great majority of the light areas are exactly the same between the two XPress runs, but a large minority are far off-diagonal.

For this reason, I would not expect H/M ratios derived from the H and L areas from those two runs to behave similarly to H/M ratios somehow calculated from a single XPress run.