Absolute Quantification in Skyline for (Peptide/Protein)

Absolute Quantification in Skyline for (Peptide/Protein) iej7  2019-10-18

I'm working with skyline (v19.1) to do some peptide based absolute quantification. All experiments are from MRM files (Instrument: 6500 QTRAP). As a general example, let's say the skyline file contains 2 proteins that are in different concentrations in my cal curve :

Protein A
-Peptide ABCDE
=Peptide FGHIJ

Protein B
-Peptide KLMN
-Peptide OPQR

I am very likely missing something basic, but don't see how to specify unique concentrations for the calibrators if Proteins A and B are in my cal curve at different concentrations. It seems like I can only specify analyte concentrations for 1 protein only and those values are repeated for all other proteins in the skyline file (i.e. Calibrator concentrations for Protein A will also be the concentrations for B).

How do I specify different concentrations for the proteins in my skyline file so that cal curve data can be evaluated appropriately?

I'm sure I'm overlooking something simple. If this is true, just point me to the right tutorial.

As always, thank you.

Nick Shulman responded:  2019-10-18
There is a value that you can set on each Peptide in the Document Grid called "Concentration Multiplier".
For most experiments where your samples are dilutions of some stock mixture, you can set it up so that the Analyte Concentration on the replicate times the Concentration Multiplier on the Peptide is equal to the actual concentration of each peptide in each sample.

It is possible that this might not be flexible enough for you. If you have spiked your proteins into your replicates in a way where the concentration of one protein is not a simple multiple of the other concentration across the replicates, then this is something that is not currently supported in Skyline.
If you need the sort of functionality where you can specify the "full matrix" of concentrations across peptides and replicates, then it would be helpful for you to send me your dataset, and I will see if I can get it implemented.

-- Nick
iej7 responded:  2019-10-19
First, I wanted to say thank you for the response. I'll get a representative file together and will upload here with further detail on what I'm looking for.
iej7 responded:  2019-10-23
Again, thank you for trying to help. The concentration multiplier may work in a pinch, but I’m wondering if there isn’t a simple solution to allow skyline to handle more information.

I’ve attached a *very* simple skyline file to further illustrate what I’d like to do. Currently, the ‘analyte concentration’ column values are repeated for both protein A and B.

1.    I have an way to easily input unique concentrations for the standards of different proteins as it seems like skyline allows copy/pasting of information into the data grid. Is there a way to simply post radio buttons in the quantitation tab asking how concentrations should be handled in the software? (i.e. Option 1 (default): Using multiplier, Option 2: Unique values (user defined))

This is probably the simplest implementation I can think of, but I completely understand if I’m asking for too much as I’m not a programmer. If I’ve said something unclear, let me know and I will try to clarify.
Thank you for your time.
Brendan MacLean responded:  2019-10-23

Let me attempt to clarify Nick's response. Most people seeking this functionality have been satisfied with using just "Analyte Concentration" and "Concentration Multiplier" in the end. The most straightforward way to do this is:

  1. Set the "Analyte Concentration" for your most concentrated sample to 1.
  2. Set the "Analyte Concentration" for all other samples to a dilution proportion, e.g. 0.5, 0.2, 0.1, 0.05, 0.2, 0.01
  3. Set the "Concentration Multiplier" for every peptide/molecule in your sample to its initial (pre-dilution) concentration

That's it. If you have initial concentrations and a dilution series this is really pretty straightforward. Are you trying for something more complicated?

iej7 responded:  2019-10-24

Maybe I misunderstood. It just seems that unique concentrations (without the multiplier) may be easier to deal with as the list of proteins one would like to quantitate increases. My thinking was simply that copy/pasting the exact values as analyte concentrations would be easier, but I can also see your point raised above about most people being satisfied with what has already been provided.

If the concentration multiplier is the best tool in skyline to accomplish that aim, I'll simply go with that.

Apologies for any confusion I may have caused and thank you for your time.

Brendan MacLean responded:  2019-11-09

When I first thought of a design for this in the early years of Skyline, long before we implemented the feature, I also thought this way and might have named the columns "Dilution Proportion" ( = "Analyte Concentration") and "Analyte Concentration" ( = "Concentration Multiplier"), but the consortium we brought together to help design the current implementation felt it was more common that researchers just wanted to define a concentration on each run and that other software had this concept of "Concentration Multiplier" to account for differences in concentration between analytes.

But, in the end, the names are just names and the concentrations are determined by multiplying "columns" (replicates) by "rows" (analytes). So, you can take either approach: 1) replicate concentrations x multiplier (default 1), 2) replicate dilution proportion x analyte starting concentration.

Sorry, if this is confusing. In the end, you will be pasting in the analyte starting concentrations, as you have suggested, but also specifying the dilution proportions.

Hope you are well past this now, but I thought I would leave a clarifying note. Thanks for your feedback.