Silac Data Dependent setting for Orbitrap Velos

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Snowcast
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Silac Data Dependent setting for Orbitrap Velos

Postby Snowcast » Wed Mar 28, 2012 12:30 am

I am just tweaking my data dependent settings for use with some Silac samples.
I understand that in the 'Global->Mass Tags' section I have to have this enabled and put in the delta masses. The samples that I will be running incorporate K6 and R8, so clearly I put in 6Da and 8Da here - but do I also have to put in combinations of these masses to take account of peptides with multiple K's/R's??

In addition in the 'Scan Event -> Mass Tags' section i have options for Neither/Low/High/Both partners. Which is appropriate to use?

I will also need to adjust my overall cycle time to keep it short enough that I ought to get a good number of MS1 scans across my chromatographic peak for decent quantification. For the last year or so, I have switched to mostly acquiring HCD data, but does the faster duty cycle of the ion trap give any advantage in the Silac scenario where you need to have a fair number of MS1 scans??

Thanks for any helpful advice,

Snowcast.

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Snowcast
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Postby Snowcast » Mon Apr 02, 2012 3:08 am

or do people just completely ignore the Mass Tags sections, acquire data on everything using standard TopN data dependent method, and let the software (MaxQuant/Proteome Discoverer) sort it out?

Craig
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Postby Craig » Mon Apr 02, 2012 8:34 am

Hi Snowcast,

I have actually never heard of anybody using this feature. One assumes that if it worked well the Mann lab and other prominent SILAC groups would be using it regularly. So with that said, and having no personal expertise with this feature, I would bet you have to enter every probable combination of K6 and R8, and probably also the m/z differences for each probable precursor charge state. I wouldn't get carried away or it might start triggering on everything and losing its specificity, so for example maybe only allow for one missed cleavage and precursors of +2 and +3. I would recommend triggering on only the light partner or the heavy partner. If it works reliably, this would (1) only take MS/MS on one partner in each SILAC pair, improving your sampling depth, and (2) allow you to do a single search with the SILAC modifications off or fixed instead of variable, which should improve your results. I think this is worth a try but keep in mind that nobody seems to use this feature so there is probably a good reason.

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Snowcast
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Postby Snowcast » Tue Apr 03, 2012 2:59 am

I put this question to Thermo tech support, and the answer was that if you use this function then you have to put in the combinations for each K6 and R8. The software can calculate the charge-states, so you do not need to input these - so your list would include: 6, 8, 12, 14, 16, 18 Da .....etc. etc. to account for multiple K's and R's .

Their advice for carrying out SILAC experiments was not to use this feature and just do MS/MS on everything using a TopN method and to let Proteome Discoverer (or equivalent software) sort it out. I'm guessing that in practice, the MassTag function is overly complex and just doesn't work well.

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Postby Kelstrup » Wed Oct 10, 2012 7:13 am

I tried it, couldn't get it to work. Anyone here succeeded?


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