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Interference or not?

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Interference or not? aulke  2017-08-22 14:53
 
Dear Skyline users.
We are having a hot discussion in the lab about data acquired by a previous lab member (see attached).
The researcher ran MRM on a peptide from partially purified protein samples (human serum). The transitions of the signal from the endogenously detected peptide look slightly different compared to the equivalent spiked-in heavy peptide.
We can detect the same peptide from the recombinant protein in a separate run.
Some more information:
The method did not contain additional transitions then the 4 ones shown.
The method did not contain additional charge stages of the same peptide.
The method did not contain additional peptides from the same protein.
The gradient was a short step-gradient. (0-30% organic in 1 minute, then 30-40 % organic over 20 mins, followed by washing)
Question to you: What do you think, interference or not? What experiments would you suggest to verify? And mostly, do you think the data is salvageable?

thanks
Anne
 
 
Brendan MacLean responded:  2017-08-22 15:25
My vote: yes, interference on y4 (and probably y6 also).

The other transitions are relatively consistent, but y4 goes from similar in abundance to y5 or essentially the least abundant to similar y8 or nearly the most abundant or a 2-4 fold change in relative peak area.

Also, worth noting that y4 as the shortest ion is the most likely to see interference.

Then I also noticed that y5 and y6 flip in their relative abundance.

Are there any other transitions you can detect for this peptide? If not, you might try a triggered MS/MS experiment to try to get a full spectrum on what is being measured, which would be expected to be chimeric, if this is interference. But, you might be able to learn more from a spectrum like that, especially if it turns out to have many large peaks that are not explained by your peptide.

Those are my thoughts. Maybe someone else will chime in with better ideas. Thanks for presenting your data on the Skyline support board.

--Brendan