How to normalize iTRAQ data

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ranio
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How to normalize iTRAQ data

Postby ranio » Mon Dec 23, 2013 8:44 pm

Hi ALL,

Recently I labeled 6 samples with iTRAQ 6-plex reagents(113-118) and pooled them together(I should have quantitated peptides of each sample before pooling or run an inject of the sample to compare the TIC intensities ), then test the samples with Q-exactive, analyzed the RAWs data with Proteome Discover. I checked the intensities of each reporter channel and found that the sum_intensities of 2 samples were lower than that of others reporters. Is there any method/ software can normalize the intensities to ensure the iTRAQ reporter signal of each channel is equal?

ranio

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Doug
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Postby Doug » Thu Jan 02, 2014 10:59 am

Ranio,

You can try simply multiplying each channel by a correction factor. The COMPASS software suite does this by default. In my experience this is fine when the differences are small. But if the intensities differ by more than 5-10% I would not recommend it. I am not aware of software that will do a more involved type of normalization nor whether that would actually work.

I highly recommend taking a small amount of each sample, after labeling, and mixing them 1:1:1:1:1:1. Then look at the summed intensities and mix the rest of your sample accordingly.

Doug
"If we knew what we were doing it wouldn't be research." -AE

Christopher
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Postby Christopher » Thu Apr 24, 2014 11:12 am

There is a nice package in R that I find provides nice normalization for iTRAQ and other quantitative data. It is discussed in this paper: http://www.ncbi.nlm.nih.gov/pubmed/20382981

Good luck!


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