Check this out! This is the same amount of the Thermo HeLa peptide mix ran on the same instrument but using two different gradients. (Click to expand)
Gradient 2 picked up 2,000 more peptides that translated into over 400 (don't check my arithmetic please, it is really early here) new proteins! Sure, they're probably single peptide hits, but I bet if you looked at them you'd see they're from low abundance (translation: cooler!) ones.
How'd the awesome scientists running this optimization get this boost? By changing their gradient from this:
2-35% B in 90 minutes
to
2-20% B in 75 minutes 20-32%B in 15 minutes.
Holy cow! That's a two thousand peptide gain? Totally for free! It looks like C-18 PepMap material (like that used in the EasySpray columns) might be a little more hydrophilic (? or something?) than other C-18 materials like C-18 Magic or older materials. Or two stage gradients are just awesome. I don't know, but if you've got the time maybe you should investigate a more shallow gradient.
P.S. Useful information:
Channel A -- 0.1%FA
Channel B -- 100% ACN 0.1%FA
Flow rate 300nL/min
200ng HeLa digest on column
QE Plus running a standard "Top10" method
Big ShoutOut to: Tara, Josh, and Lani cause they did the work. I just stole from their slides!
Ben,
ReplyDeleteCan you suggest a training resource which could help me figure out if my MS2 is optimal? I feel like i am not getting good ID's because of crappy fragmentation (for example my precursors are around 1E5 and i'm getting 1e3 for fragments). Also, could you recommend a good site to help me figure out if my PD 1.4 workflow is optimal?
Thanks
Cliff
Cliff, Are you looking at CID or HCD spectra? This is short, but for HCD spectra this is kind of the rules that I use: http://proteomicsnews.blogspot.com/2013/11/what-does-good-tmt-or-itraq-msms.html
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In general, though, you want to see an almost complete loss of your parent ion (in HCD, I gear toward 5-10% of base peak being my parent ion. In CID, I'd rather see <1% of base peak being parent...in theory, I think it should be completely gone.) And you should see a loss in total signal that makes sense. For example, if you are triggering something at 1e5 resulting fragments of 1e3 make sense if you have hundreds a very right fragmentation pattern (lots of peaks). It should be somewhat cumulative. Now. Please keep in mind that every time a packet of ions is transferred there will be loss. If I have 1e5 ions in my ion trap and I isolate them, if I transfer them to the Orbitrap, not every ion is going to get there. According to people I know on the Fusion Tribrid development team, one of the biggest reasons that instrument seems more sensitive is that it does a better job of routing ions from one place to another than other systems. If you look at an Orbitrap XL, the first one I used, there was a big loss from the ion trap to the Orbitrap...and an even bigger loss from the trap to the HCD cell and back to the Orbi. I think you could see almost 2 orders loss in signal just moving ions into the HCD cell with no CE and back to the Orbi. Later generations of the HCD cell were MUCH better and many were retrograded to the Orbi Velos cell.
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PD 1.4 workflow? You could always send it to pd.support@thermofisher.com and get their feedback. They should be able to look it over quickly and give you advice. You could also post a text version to the Thermo Omics portal if you're a registered member. Heck, you could send me the text version (orsburn@vt.edu) and I might have time to take a look at it on the weekend (can't guarantee it, I get kinda swamped, but I'd give it a try! and I'm snowed in!)
What are the result filters that were applied here or what are the result filters that you typically use?
ReplyDeleteThanks
cliff
Howdy!
DeleteIf I remember correctly, this is processed with 10ppm MS1 tolerance, 0.02Da MS/MS tolerance, static Carbamiodomethyl on C, variable Met Oxidation and percolator with default cutoffs and a "high" confidence filter. That was generally the default processing method for PD 1.4