Hi,
I am currently doing data analysis on a proteomic dataset and am attempting to compare a set of protein markers between different methods of sample preparation. However, due to the various limitations I am unable to use any form of relative abundance, and are therefore trying to find an alternative method to compare the different sample preparation methods.
My current idea is to use IBAQ intensities and compare the relative expression between sample preparation methods. To do this I want to calculate the relative expression of certain protein markers within each sample, before comparing this relative expression between the methods.
To give you a better understanding of my dataset, here is a brief summary of my experimental setup:
Experimental setup
* I am comparing four different sample preparation methods (ie, method 1, 2, 3 & 4).
* There are three unique biological samples/replicates (3 different controls) in each sample preparation method (ie. 12 samples).
* The sample preparation methods are performed as technical duplicates, meaning there is an A and a B series (ie. A total of 22 samples).
How I want to use IBAQ
Now, specifically, what I want to do is to compare the different sample preparation methods using a set of protein markers and see whether these markers have increased or decreased expression based on the method used. My idea when using IBAQ is to calculate the IBAQ percentage expression of each marker compared to the total IBAQ intensity of the same sample.
Somewhat like this:
Serum albumin (HSA)
Method 1: Total IBAQ = 10 - Total HSA = 1. Percentage of expression: 10%
Method 2: Total IBAQ = 7 - Total HSA = 3. Percentage of expression: 42%
Method 3: Total IBAQ = 20 - Total HSA = 1. Percentage of expression: 5%
Method 4: Total IBAQ = 120 - Total HSA = 50. Percentage of expression: 41%
(I plan to average the percentage of both technical and biological replicates).
My question
My question would then be if: based on these calculation I can conclude that Method 2 contains the most amount of HSA while method 3 contains the least?
All help is appreciated!
Hi Anders,
iBAQ itself is already an estimate for absolute quantification. So there is, in my opinion, no need to normalize to the complete IBAQ intensity I think it might give the wrong impression.
Picture for example you would simply have HSA, digest it and measure it, lets say 2 µg. If you compare that to your analysis you probably would get 0% since the complete intensity is actually coming from HSA. Or am I getting something wrong? Also then, calculations would depend highly on your "background". With that I mean all other proteins but HSA.
Since the iBAQ intensitiy does not rely on the the whole protein distribution (like LFQ) it will be fair to compare simply the iBAQ intensities. Note that saying does not rely on your background is probably not entirely true from an instrument speaking point. Because iBAQ checks for identified peptides. If your background is covering all "peptides" from your marker you might get different values for the iBAQ intensity due to the background. So keep that in mind.
Hope this helps
Hendrik
Hi, Henrik! Again, thank you for the response.
I might not have been clear enough in my explanation, so allow me to rephrase the question.
Let’s say I have two samples where i want to figure out which method that contains the highest percentage of HSA of the two.
How I would like to do this is by taking the IBAQ value for HSA for sample 1 and comparing this to total IBAQ level of all proteins in sample 1 (ie. sample 1 background IBAQ). Then I would compare this "ratio" to the same "ratio" calculation done with sample 2. So what I'm basically doing is treating each sample as seperate and only compare the said "ratio" between samples.
Best regards
Anders
Hi,
then your method looks very fine. You can also test this with the "normal" measured intensity and you should not get very different values.
Best greetings
Hendrik
P.S for biological and technical replicates - you should pool your technical replicates before comparing biological ones.
Hi Hendrik,
While I did end up using this method, I ended up doing some testing prior to using it, as I after asking this question, found out that the standard/optimal IBAQ value is calculated based on logarithmic fitting against a UPS protein standard spike in. In my present experiment, I use no such UPS spike in.
So what I actually ended up doing was to test the correlation between UPS spike in IBAQ values against "Total Protein Approach method (incl. total protein & protein concentration)" and my IBAQ values without UPS spike in. Here I found that the best correlation was seen when using IBAQ values w.o. UPS, which I subsequently used.
And again, thank you for the assistance.
Best regards
Anders
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