September 3, 2011 at 1:16 am #1359KelleyParticipant
I need help with ideas to solve this issue for a fellow researcher…
Pawel wanted me to shoot you an email regarding some issues I’m having with pulling the TempoLC and MALDI together for iTRAQ. Melinda is aware of this issue as she watched part of my acquisition yesterday. Brief background:
We use TempoLC to fractionate our iTRAQ samples into 140 spots/fraction. We have total 12 fractions = 1680 spots, requiring 2 MALDI plates. We upload the plate design from Tempo to MALDI, so we can do a batch acquisition for 2 plates and the MALDI knows where are spots are located. The problem is that the laser doesn’t always hit a spot on the plate. This gives poor results and yesterday’s run gave me the worst ever (and Melinda can testify to how bad the laser was missing the spots). From 1680 spots I only got 550 MS/MS acquistions. This is crappy. The most I got was 6900 MS/MS from 1680 spots (prior to recent PM), on average we (between Gwen and I) tend to get 1500-2500 MS/MS. Melinda did suggest a “center bias” instead of “uniform” for laser shots and I have not tried this yet. So this is an option.
iTRAQ is a proteomics platform we want fully functional here, so I think Pawel wants to see if I can get the core help to figure out what is going on.
I emailed Gwen (who’s iTRAQ platform I’ve been using) and asked him for suggestions. Here is what he said:
“the tempo will always save the position of the spot the same way. In fact, the depositor tip will alway be placed at the same position above the plate and if the plate is slightly moved it wont change a lot in the MALDI. Now you can do 3 things:
1: You can go to the MALDI with your plate, you randomly choose 5 or more spots on the plate and you shoot. Then you can watch if the shooting is manly on the spot or not. I would recommend to choose spots in the middle of your run, this way you’ll see how good are the spectra and how dense is the spotting and also if the intensity of your spectra are good enough. To be even more careful, you can ask to show the intensity and the resolution of the peak (Spectrum>label>check boxes), if you see that resolution is too low (around 100s for instance) there might be an issue (this we can try at anytime)
2: You put a plate in the TEMPO, you ask it to go to position A1 above the plate, and check if the depositor tip is in the middle of the circle on the plate. If not this position need to be calibrated, I never had to do it, if you have to do it I think you had to “read the book”, :o/ sorry (The Tempo is calibrated because on all my plates, the spot is directly on A1)
3: you can calibrate your first plate in the MALDI to make sure that it shoots at the right place. this is the worst scenario you can expect and I give you the idea but I don’t recommend it because a calibration for 1 plate may not be the same for the next plate so… ”
So somehow we have to make this automated as there is no feasible way to do it manually. I have some plates we can practice on — the 2 plates I ran yesterday I think we can shoot some more — I”m not sure much sample/matrix was used up. If these plates aren’t good enough, we can easily spot some new samples and work with that.
I have 4 more MALDI plates ready to shoot, but I will not do so until we can be sure that everything is smooth. I can’t lose any more samples. What do you think our next step should be? Should you, Melinda, Pawel and I sit down next week and see if we can put together a plan of attack? Let me know.
THANKS FOR ANY IDEAS YOU HAVE TO OFFER!!!
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