[Sticky] Problem in how to use PepCalmix peptides result in SWATH RT calculation
I had run several SWATH experiments with PepCalmix spike as an internal standard for later rentention time calibration or adjustment during SWATH quantitation. However, I found problem in adding the PepCalmix information in Ion Library generation at the beginning of SWATH quantitation so I cannot pick any reference peptide for RT calculation. Besides, I was confused by the inconsistent of PepCalmix reference peptides picking results from all SWATH experiments. If I am not wrong, it is supposed there would be no great different of the reference peptides signal among the PepCalmix spiked samples for SWATH experiments. What is the possible problem if the inconsistent signal of reference peptides occuring? The TIC for those SWATH experiments were well aligned and overlapped.
I used the spectral library that was provided with the SWATH QC protocol from Sciex. I then copied and pasted the pepcalmix Q1/Q3 ion and library information into my own spectral library prior to alignment so I ended up with my own library with the pepcalmix ions included. Edit: I obviously put in the real retention times of the pepcalmix ions. I did it this way to save the time re-searching the wiff file with pepcalmix peptides in the fasta.
First of all, when adding PepCalMix to your ion library, you need to make sure you have the PepCalMix in the same retention time frame as your main ion library. Please see this post on Adding PepCalMix to your Ion Library.
Next, assuming the RT frame is correct, you might see some varaibility in peptide selection for the PepCalMix as the retention time calibration peptides depending on matrix. The included fragments in the general library work great in human cell lines, but yes you can adjust these if you are finding other peaks getting selected in other matrics. Do this by removing ions (probably start with the lower m/z b/y ions) and checking to see if the correct peak is selected. Then you can save your Ion library with the subset of fragment ions that work well for this matrix.
When spiking the PepCalMix into your real samples for RT calibration, and if everything is set up correctly, then the signal variation will come mainly from pipetting. If you are seeing more variability, then something might not be set up quite right. Do you think this is coming from mis-selection of peaks, adjusting the fragment ions as mentioned above might help.
Thanks. I will try.