skyline and quantitation by MS1 XIC filtering on agilent or other QTOF after DDA ms/m

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congriver
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skyline and quantitation by MS1 XIC filtering on agilent or other QTOF after DDA ms/m

Postby congriver » Thu Sep 19, 2013 2:44 am

Hi
For my first ever protein quantitation experiment I plan to try the simplest approach ie label free quantitation.
I am considering using skylines recently added quantitative capability using MS1 XIC( ie.not MRM) for label free protein quantitation on an Agilent QTOF to quantitate differences in proteins between samples. I was wondering if anyone had any advice or tips before i start. I'll be doing identification using xtandem and or Peaks . The only quantitative software( I'm a biologist not a computer scientist) I have access to is Peaks which does have a basic quantitative capability Peaks but it's probably not sufficiently rigorous for this type of work.My plan is to compare Skyline MS1 quantitative filtering to Peaks quantitative software.So any tips for the skyline approach.
thanks
Brendan

tsbatth
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Postby tsbatth » Thu Sep 19, 2013 9:16 am

congriver wrote:Hi
For my first ever protein quantitation experiment I plan to try the simplest approach ie label free quantitation.
I am considering using skylines recently added quantitative capability using MS1 XIC( ie.not MRM) for label free protein quantitation on an Agilent QTOF to quantitate differences in proteins between samples. I was wondering if anyone had any advice or tips before i start. I'll be doing identification using xtandem and or Peaks . The only quantitative software( I'm a biologist not a computer scientist) I have access to is Peaks which does have a basic quantitative capability Peaks but it's probably not sufficiently rigorous for this type of work.My plan is to compare Skyline MS1 quantitative filtering to Peaks quantitative software.So any tips for the skyline approach.
thanks
Brendan


I recommend Maxquant, it does quantitation based on MS1 and identification, but honestly I would recommend against it unless you're doing high res MS (>50k), also it depends on your organism. If you're working with human peptide sample mixture is a lot more complex then say compared to bacteria. What is the context of your experiment ?

JDRCHEM
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Postby JDRCHEM » Fri Sep 20, 2013 6:52 am

I've used a number of different software packages for label-free quantification based upon extracted ion chromatograms and/or spectral counts. All of them work well enough if you are willing to devote enough time into learning how they work. I will be honest, I do not like putting effort into learning software unless I know I will use it frequently. It is challenging for programmers to make a software package that balances functionality with ease-of-use and I get easily frustrated by software that does a million things but very few of them well. That being said, I really like using Skyline for MS1 quant. Pros: fast, intuitive, vendor neutral, great technical support, and free; Cons: it lacks advanced visualization tools and reporting features that commercial packages often have.

I haven't attempted to quantify thousands of peptides using Skyline. But, I have built spectral libraries containing 10 to a few hundred peptides to process up to 128 raw files (all at once) and I am very pleased with the results. The peak picking algorithm works really well, but there are occasions when I have to go in and manually integrate a peak, but that is rare enough that it doesn't bother me. When my chromatography is good (reproducible retention times) I find that to peak picking is almost flawless.

You can search the Skyline user forum and their website to see if the current version of Skyline works with PEAKS, but their targeted method editing tutorial indicates it is compatible with X!Tandem.

-Jason

brendanx
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Postby brendanx » Sat Sep 21, 2013 12:19 am

Thanks for the positive review, Jason! I would be interested to hear more offline about those cons, as the Skyline Team and I are always working hard to improve the software. It is always good to know what people want most.

Coincidentally, we have recently been in touch with the PEAKS team. They approached us requesting support for integrating with Skyline spectral library building, and I am happy to say that we just finished testing the support they are providing, which involves exporting pepXML and mzXML that can be correctly built into a spectral library, containing spectrum source files and retention time information, for use in MS1 filtering experiments. Contact them about availability in their next release. We worked with Ron Beavis last year on providing spectrum source file information in native X! Tandem results XML, though that may require an explicit setting in your X! Tandem parameters. Check with GPM support on that. There are people using X! Tandem successfully for MS1 filtering experiments.

And, of course, as Jason notes, Skyline supports TOF and ion trap instruments from Agilent, AB SCIEX, Bruker, Thermo and Waters. So, you should be able to get it to work with your instrumentation.

As for being a "biologist not a computer scientist", I have always maintained that my target software expertise for Skyline is people comfortable using Excel and Word. Feedback over the years has encouraged me to believe I am meeting that goal. One mass spectrometry PI told me this summer that she teaches her biologist collaborators how to use Skyline and then lets them process their own mass spec data that she is collecting for them.

I hope you will try and enjoy using Skyline. The support board (Help > Support from within Skyline) is free, if you run into problems getting started, but I have also receive a lot of positive feedback on the documentation.

Good luck, and thanks for your interest in using Skyline for your research.


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