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Crowdsourcing: Optimized Rolling Collision Energy Curves for ID and SWATH Acquisition

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This video describes a short optimization project to measure the optimal collision energy curves for TripleTOF systems, for performing IDA and SWATH experiments. Rolling collision energy curves were measured on 3 different instruments and then validated on additional instruments. Gains of 5-10% in peptide/protein IDs were found with the new curves. The new curves are compatible with the 100 variable window SWATH method and the Collision Energy Spread (CES) observations are already built into this VW method.

We’d like you to give these new settings a try! You can download the text file or view the table below for the new settings. Enter these values into the IDA CE Parameters Table found under the Scripts menu. 

CrowdSourcing OldvsNew

The next time you're doing an IDA or SWATH experiment, please try these new collision energy curves and compare them to previous results. Let us know if you see more confident IDs or better quality quantitation. If you have questions on how to do this, please ask!

TripleTOF CE Curve.txt

This topic was modified 5 months ago by christie.hunter@sciex.com
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Topic starter Posted : October 22, 2015 7:05 pm

"If you have questions on how to do this, please ask!"..

... How do you do this please? 😉

Where does the "tripletof CE curve.txt" file go in Analyst?

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Posted : October 23, 2015 9:58 am
christie.hunter@sciex.com
Member Admin

Thanks for the question David.

In order to change the collision energy equations within Analyst, go to the Scripts menu and select IDA CE Parameters. This opens up the window shown here, where you can type in the new values for each charge state equation.

Remember that these CE values are taken at the time of acquisition, so to switch back and forth to test the CE values, you must make the change before you submit your batch. Then change to the next values right before you submit your next batch.

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Posted : October 30, 2015 7:18 am
Roz Jenkins
Member

Hi Christie,

Two things. Firstly, are the suggested new equations appropriate for both the 5600 and 6600? Secondly, the values included in the table in the video are not quite the same as those included in your response to David. It's only the equations for the unknown and +1 charge states that are different, but it would be useful to get the best parameter set available.

Many thanks,

Roz

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Posted : November 13, 2015 10:59 am

We've been using the new settings on the 6600 for a little while now after running some comparisons.  Initial results are that the new settings are definitely "better" in IDA - as difficult as it is to quantify a stochastic process!  Based on the number of proteins, peptides and spectra at 1% FDR as reported by ProteinPilot 5 I'd say consistently a 3-4% improvement in the numbers.

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Posted : November 13, 2015 11:21 am
christie.hunter@sciex.com
Member Admin

Great to hear that the new equations are providing a boost in ID quality, David!!

Roz, the equations will work for 5600 and 6600, we collected the data on both instruments and the curves were very close, close enough to go out with a single set of curves. Sorry about the discrepancy with the table, I noticed after we recorded the video that I hadn't updated the numbers for the unknown and 1+ equations. We always set the unknown equation to be the same as the 2+ ion. Note that we didn't measure the curve for the 1+ peptides, so this is a guesstimate at this point based on the trending of the other charge states.

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Posted : November 13, 2015 11:30 am
Roz Jenkins
Member

Thanks David, I'll definitely give them a go. BTW, do you have any issues with source contamination on the 6600? I run the default curtain gas at 25 but I'm thinking of upping it.

Thanks again,

Roz

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Posted : November 13, 2015 12:03 pm
Roz Jenkins
Member

Thanks Christie, I'll give them a go next week and report back.

Have a great weekend.

Roz

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Posted : November 13, 2015 12:04 pm
Roz Jenkins
Member

I'm really struggling with contamination on my Triple TOF 6600. If I run a SWATH batch, the instrument needs cleaning every single week and even during the course of the batch, I can see my MS/MS signal dropping. Surely, that can't be right? I have my curtain gas set to 35 to try to help, but this issue is beginning to make the system unuseable. I'd appreciate some suggestions (other than conversion to micro flow, which is not possible just now). 

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Posted : August 25, 2016 6:30 am

While we're running microflow exclusively nowadays, I can't comment specifically - however a while back we had issues of dropping sensitivity among some other issues and the engineer cleaned the instrument ion path using a new protocol/kit (not alconox).  Now I'm not saying that was the problem/solution as we had other things looked at at the same time, but since then the sensitivity has been much more stable (829 tuning peak was staying about the same week after week without cleaning - and at intensities at least 4-5 times higher than it had been over the first year).  The impression was that after that clean, contamination was much slower than it had been previously.  But then with nano, as you know, you're spraying that much more directly in...

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Posted : August 25, 2016 9:21 am
Roz Jenkins
Member

Hi David. Many thanks for this. I understand you also spoke to the lovely James - he worked with you while you were doing your PhD, didn't he? Rob Lane mentioned the new cleaning protocol, but didn't get back to me with the details. Do you have a protocol you could send me? If not, I'll badger Rob. He hasn't heard from me for at least 3 weeks, so he must be missing me by now! Thanks again, Roz

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Posted : August 25, 2016 10:22 am
Melody
Member

Dear all,

I failed to change the CE parameters. Any suggestion about this error?

Thanks a lot!

Best regards,

Melody Lam

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Posted : April 12, 2017 6:31 am
Melody
Member

Problem solved!

I just find out that the parameter file at 

[Drive:]\Analyst Data\Projects\API Instrument\Preferences\IDA CE Parameters.txt

has been marked as read only. 

After removing the "read-only" status, I can change the CE parameters.

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Posted : April 18, 2017 1:05 am
christie.hunter@sciex.com
Member Admin

Thanks for figuring it out and sharing!  We figured it had something to do with Windows user permissions or Analyst user permissions. 

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Posted : April 19, 2017 10:56 am

I notice the values in the file attached above, the video and screenshots are not the same as the later post here:   https://sciex.com/support/knowledge-base-articles/questions-about-optimal-ce-voltages-for-peptide-analysis-on-tripletof-systems

I assume there's been some iterative improvements again and I should use the newer values from Jan 2018 now?  Perhaps the .txt attachment should be updated if this is the case?

As a side note - I checked my CE parameters (I'd changed them some time ago to the optimised ones), and they had reset to the Analyst default values - possibly at a software reinstall/update during an engineer visit.  I've no idea how long they've been like this, so its worth checking periodically.

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Posted : March 29, 2018 6:16 am
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