Crowdsourcing: Optimized Rolling Collision Energy Curves for ID and SWATH Acquisition
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.
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!
"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?
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.
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.
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.
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.