Wednesday, March 9, 2016
Blast from the past -- Observable peptides!
I'm giving a couple of talks today and I was digging through my references.
In 2011, this team in this paper found that in a normal run on 2011's high end instrumentation, they could see via MS1 that there were >100,000 peptides (or features) available in their samples. They also found that they could only ID about 16k of them.
Funny thought here...I have a Lumos RAW file open and in front of me and I've got >100,000 individual fragmentation scans that were obtained on a 2 hour gradient. The instrument was set to ignore isotopes and make exclusions based on the uncharged mass, so even if it saw a +2 and +3 precursor of the same peptide, it ignored all but the single most intense (if it was the +2, it will not fragment the +3! Yay for tons of onboard processing power!!!) So...I should really have fragmented 100,000 unique things in this run.
Interestingly, even if I run this file with multiple alogorthims using tons of possible PTMs, alternative cleavages, a seriously awesome database, export the unmatched spectra for de novo sequencing with the awesome DeNovoGUI and combine everything with something as close to a 1% FDR as I can get, I still only get about 40% of them matched out.
What the heck is the rest of this stuff?? They sure look like peptides from their isotopic distributions, they stick to C18 and elute like peptides....seems like there are some HUGE components of the human proteome that we don't know about yet (or maybe tons of little things?!?). Either way, I'd totally appreciate it if somebody would figure out what that other stuff is just so I'd know. If it is really a major biomolecule class we don't know about, maybe you can collect a Nobel for your troubles.