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.
de novo sequencing with tons of computing power
ReplyDeleteDear Ben,
ReplyDeleteHonestly, I'm not so convinced by data collected using UM Method in the Lumos.
I noticed several time that the ID rate is clearly better using HF instrument. The number of collected spectra is lower but the overall number of unique peptide looks better.
For DDA analysis using a relatively high number of peptide (300 ng of Hela cell lysate from Pierce in a 2 hours gradient ), HF seems to be better compared to Lumos.
Moreover, I expect that High resolution should be better for denovo sequencing compared to high low resolution in the Lumos.
Do you have the same feeling since you have access to such kind of raw data ?
Best,
Howdy,
DeleteYeah, the Lumos does achieve more MS/MS scans when running the ion trap simultaneously, and I'm probably in your camp. While I see the value still present in low res MS/MS scans, I definitely prefer Orbitrap MS/MS. What is interesting, though is that I have a FT/FT dataset from this same sample injection where I just flipped the Lumos over to Orbitrap MS/MS and, you know what, it isn't that many less MS/MS scans. Many people I know who have Fusion/Lumos instruments don't use the ion trap at all. They treat their Fusion's like Q Exactives with the added benefit of MS3 TMT/iTRAQ quan and ETD. (Still gotta have an IT for ETD for now!)
Dear test test
DeleteI have an OT Fusion. My first goal was to define the best way to optimize the protein identification ... and I think exactly the opposite of what is written. The HCD IT mode exceeds without any shadow the HCD OT mode in terms of sequenced peptides. The reason? IT is significantly more sensitive to the OT and the ability to parallelize the processes provides a scan of unquestionable advantage.
this is true if my only goal is to maximize the number scans and identifications and high resolution (for Denovo studies) is not my interest.
Dear Ben,
DeleteI agree that LUMOS/FUSION are quite versatile tools and they enable many great acquisition modes.
In order to have my own advice on data collected with the 2 instruments, I was wondering If you can share some raw files from LUMOS and QexHF with identical LC separation in order to have a fair comparison ?
Best regards,
have you tried to look at "High Performance Mass Spectrometry and Informatics for Chemical and Biological Exploration of the Proteome Prof. Bernhard Küster" ?http://info1.thermoscientific.com/proteomics-leadership, maybe the answer is there.
ReplyDeletehowever good point ... to reflect!
Andrea
Andrea,
DeleteNo, I haven't checked that out. I will bookmark it. Thanks for the link!!!