Hi Frank,

I think people usually get the template from their own instrument control software, though I admit I'm unclear on how that works since I'm not an actual mass spec operator.

I'd say contact your local sales engineer for advice, or perhaps someone else will chime in here with a more informed answer.

Best,

Brian Pratt

The template really is system specific and should be created on your instrument PC. It is the method file as you would create it minus the quadrupole (precursor m/z) and time (time and window) coordinates that Skyline will provide. Imagine you were setting up a simple single precursor m/z PRM method on your Fusion and create the method file for that, but leave out even the one target. You need a template that has you tune and LC conditions.

Hope this helps. Otherwise, I have passed along this thread one of our contacts at Thermo, who may be able to give you a clearer answer.

--Brendan

Hi Frank,

What Brendan suggested is correct. Generate a .meth file in XCalibur Method Editor that has all of your LC and MS tune conditions (source conditions) and PRM mode selected. I usually save this kind of file (that doesn't have the inclusion list populated as "method_details_TEMPLATE.meth", where "method_details" describes the method in the filename, and "TEMPLATE" denotes it doesn't have an inclusion list populated, but is instead a template for Skyline to work with. If you're using the same LC gradient as what you used in your DDA runs, you could open that .meth file and change the MS experiment to "PRM", this way you know the gradient is preserved.

Remember, if you have a lot of peptides you are targeting at once, the MS will try to acquire them no matter how many there are or how long it takes. You may have to adjust your max fill time or keep an eye on how many concurrent peptides are eluting to make sure you will be getting enough points across your chromatographic peaks.

When you are in Skyline and want to export a PRM method, this TEMPLATE.meth file you generated is what you should select...Skyline should be able to access it and populate the inclusion list with your m/z, z, RT details for the PRM mode method.

If you need any help, please let me know!
Sue

Dear Brian,Brendan and Sue,

thanks very for your suggestions. I will try this and see how I get on. With Sue's detailed instructions I should be able to get this going. I'll check the the points per peak. Does Xcalibur tell you this? I'm more familiar with setting up SRM methods on Masslynx on a Water's instrument. there I aim for minimum of 9 points per peak.

I'm currently setting method up with the AGC target at 5 e4, 'injection ions for allavailable parallelizable time' selected, maximum injection time at 54ms, with resolution at 30000 and scan range 100-2000. I'm aiming to target about 250 masses over a 143 min gradient. Window around the RT is 3 min on either side. Any advice on these settings would be appreciated

Frank

Hi Frank,

If you open your .raw file in XCalibur, you can change the display type to "stick" by right clicking in the chromatogram (make sure the pin is green), select "display options", then under the "Style" tab, click "stick". This will show you where each data point is and you can count them to determine the points across your peak.

I'm pretty sure Skyline has a way to report the number of points across the peak in the results grid, but I'm not 100% sure how to do that.

What are the widths at the base of your chromatographic peaks? You want to shoot for a minimum of 8 points across the peaks, so you can figure out how long the instrument cycle time based on this. You may have to look up in the MS user manual how long it takes to acquire at 30K to see how many PRM targets you can get without going past your calculated cycle time.

Best,
Sue

Yes. Skyline has a Points Across Peak value available for use in reports and the document grid. It is under Transition Results, but if you are in the report or document grid customization form, you can click on the Find (binoculars icon) button and type "Points" and click OK to find the field.

For more information about customizing reports and the document grid, see the "Custom and Live Reports" tutorial:

https://skyline.ms/tutorial_custom_reports.url

Hope this helps. Thanks for jumping in, Sue.

--Brendan