Tuesday, July 8, 2014
SIEVE or PD for label free quan
In the context of obtaining peptide IDs, nothing can aid you the way good chromatography does. More and more, I'm seeing that this is paramount with today's super fast instruments.
What about for assigning quantitative data? How important is my chromatographic alignment? SIEVE is a program that puts chromatography up front -- data in tight m/z windows within a small retention time windows are chosen as "frames" and those are what you do your quan on.
Proteome Discoverer has a label free quan node - the "Precuror Ion Area Detector". When used in conjunction with an event detector, you can pull out ions from your files within a narrow ppm range (maximum is 4ppm! I use 2ppm) and compare those peptides. Retention time is never considered in this current iteration.
This is how I've always done label free quan. Mostly because my previous employers did not purchase SIEVE. I'd manually export my label free quan data and use a short script in DigDB to remove matching peptides that did not match in retention time (or pull out the PSMs with the list the retention times, subtract them and throw out anything on the outside of my retention time window, then recompile the report).
Yes, it is a lot of work, and essentially a work-around. But if you can't afford another software package, you have a backup plan. You can follow a link here to a video I made regarding setting up PD for label free quan.