Monday, December 30, 2019
Optimizing/reducing in source fragmentation on 3 different Orbitrap systems!!
Talk about a useful study! If you're doing tryptic peptides, maybe this isn't all that useful, but if you are working on anything that is more fragile than that (glycopeptides? PARPylated? intact/native, metabolites...we could go on an on here) this is probably worth at least thinking about.
On the letterbox systems (the ion tranfer tubes with the great big rectangular holes) we use lower RF% to start out with. For peptides on a Q Exactive or HF system, I typically err toward an RF of 50-60%. On the Lumos or Exploris we're typically doing 40-45% for peptides.
The great Katie Southwick explained RF% to me years ago (I need an ELI5 once in a while) as the amount of pulling force in through the very front of the instrument. Bigger things probably want a higher %RF but you have to keep in mind that there are downsides to that extra force and you could break apart smaller or more fragile things.
In this study, this group takes some of the more fragile things that we all hate to work on -- lipids -- and painstakingly compare different systems with different source conditions.
The chart at the top is the one I find the clearest and most valuable out of this great study -- when I'm looking at something that is clearly fragile and I've looked at it on whatever instrument is available -- this provides some guidelines for normalizing a setting that I probably didn't pay enough attention to.