How to do quantification of reporter ion quan replicates in PD 2.2

Thank, Dr. K for this first great PD 2.2 question that Dr. P didn't cover in his/her videos!

Here is the question: If you have an experiment like this reporter ion one above where your TMT-10plex set has replicates within it's set, how do you set that up so that PD 2.2 knows these are replicates and will allow you do get p-Values and use Volcano plots?

First off, you're going to need 2 study factors -- one for your samples and one for your replicates. Something like this will work for the example above

Here I've set up 5 conditions for my TMT-10plex control runs I did with a friend in Boston a few years ago. This is human cell digest added in a ratio of 1:2:4:8 with 2 replicates of each (and 4 1:1).

Next, I'll have to set up the Samples tab so they know which is which. Something like this

I just called the replicates 1 and 2 in order for simplicity sake.

Now, when I go to the Grouping and Quantification tab, when I select my conditions, this "Nested Design" wording pops up and my samples look like this:

DISCLAIMER: (Maybe this should be at the top) I don't know if this is how you do this. There is probably a better way. In this example it looks like the ratio of B1/A1 is obtained and then compared to the B2/A2. Works for me for this example, but please only take this as a way to get started!

What do the results look like? Spot on!

I'll have to look at the RAW file again, cause it looks like A and E are the 1:1 (and we had a bit of variance due to our pipette accuracy at 1uL.) but the other channels look right and E/A looks about 1:1

Now that we have replicates then we're allowed by PD to create Volcano plots to find what is statistically and quantitatively interesting. They should look like this (from a Compound Discoverer set we're working on):

However, if ALL your peptides are 1:1 or 1:2...that doesn't work very well...the 1:1 sample looks like this:

(...everything is at/around 1...I made this even messier by just processing peptides in a very narrow m/z range so that I could do several iterations before I had to go to work)

Follow-up question: Where are the p-Values coming from in the Volcano plot?

They are the -Log10 (unadjusted) Abundance ratio p-Values for the peptide/protein.

By default template you probably only see the Adjusted p-Values. You can unhide this value by clicking the Field Chooser (1) and then checking (2) above.

If you've also forgotten all of high school (Logs?), the way to convert the Abundance Ratio P-Value in the sample output to the one in the Volcano plot in Excel is something like "=ABS(LOG10(value))". Took me a couple tries to get it right and the numbers match.