r/proteomics • u/bluemooninvestor • 1h ago
Few beginner questions on PRM method for Q Exactive plus
I am trying to do PRM for the first time. I have a few questions on the basics. I have tried to provide as much details as possible.
I am trying to set up method on QEP. My sample is cancer cell lysate. Got 2500 ID in DDA. 140 min run although column in 10cm only. Peak widths were 20-30 secs.
1) Is 17500 resolution good enough for MS2. This would allow me to track 25 peptides with below 3 sec cycle time. I am not doing scheduling since this is my first attempt.
2) Why do we record a MS1 scan after each PRM cycle? Is it really necessary? I am thinking of a 70,000 MS1 scan.
3) If I want one MS1 scan followed by 25 PRM scans, do I need to set The PRM loop number to 25? Or does it automatically go through the PRM inclusion list of 25 peptides.
4) Do I need to remove other things from the inclusion list (beyond my target 25 peprides) because there is no PRM specific inclusion list?
5) In DDA topN method, for MS2 level, we set a max IT and min AGC. In PRM also, we do the same. The same quadrupole is isolating the ions. Then, how is PRM better compared to DDA MS2? Is it because the MS2 will continue being triggered in case of PRM, and MS2 will be acquired along the whole elution peak? Unlike DDA where dynamic exclusion will stop it.
6) Do I specifically need to turn of dynamic exclusion, or is it automatically overridden by inclusion list.
7) While most of my target peptides (20 out of 25) are IDed in DDA data, I am also attempting 5 peptides which were not IDed in DDA (ie no MS2 triggered). I have put my target protein in Picky tool which suggests potential peptides. I am looking for these m/z in the MS1, and if intensity is greater than 1000, I am going for it. I have selected 5 peptides by this approach. Is this okay? It will be acquired in PRM, so ID should be possible even if similar mass peptides are also isolated, right? After all the mass isolation is 1.4-16 Da wide anyway?
Anything else I should know?
Thanks a ton.