Wednesday, September 6, 2017
PROCAL -- QC for retention time AND collision energy!!
If you are using HCD fragmentation for proteomics, I recommend you stop what you are doing right now and check out this awesome new paper just acceped at Proteomics!
Yes, I'm talking to you! (This pug on loop is cracking me up. Hard.to.type!)
Seriously, I've been looking for something like this resource for years! Chances are you have been as well. I've tried answering the question "What does a good HCD spectra look like?" a couple times on the blog. But these rules I've been using to troubleshoot fragmentation are imperfect at best -- and wrong at worst.
PROCAL -- is a resource that 1) Improves upon the PRTC idea (15 peptide standards? -- how about 40 retention time standards!) but goes a whole lot further.
The peptides are not heavy labeled -- their sequences don't exist in ANY protein on earth -- this allows the standards to be integrated into your proteomic processing workflow without any crummy work-arounds -- except adding the peptide sequences into your FASTA for quick identification checks (AWESOME!!)
The retention time calculations are even better with PROCAL -- several peptides were picked on purpose that have extremely similar retention times. This allows extremely sensitive monitoring of column performance.
Okay -- this is all super cool -- BUT -- they study how these peptides fragment under HCD in different instruments -- allowing the HCD collision energy to be calibrated between platforms!! I'm fuzzy on the details and I'll have to come back to this later, but it looks like they used Skyline transition intensities to do the measurements.
I've got to run, but not before I start downloading these RAW files from PRIDE / ProteomeXchange.
Nuts. The paper isn't proofed yet, but when it is the RAW files are available here.
Gotta get out the door, but I'm so inspired that such a huge technical hurdle has been addressed for all of us!