SILAC quantification with targeted SIM

[Will Comstock]

Hi all,

I'm trying to use tSIM-ddMS2 to identify specific peptides in my sample, but I also want SILAC quantification of those peptides using XPRESS. I've made sure the MS1 isolation window is wide enough to see both Light and Heavy peptide peaks for everything in my inclusion list (30 daltons, so my heavy peptides which are +8 or +10 are detected). However, when I search with Comet and XPRESS, there is no quantitative ratio provided, just -1.0. XPRESS appears to be looking in the wrong place for the light and heavy peptides, but I am not sure why or how to redirect it to the right parts of my MS1 spectra. If I look at the precursor scans manually via QualBrowser, both peaks are definitely being detected.

Has anyone here tried using XPRESS to do SILAC quantitation in targeted runs before? 

Here is a folder with my data, search parameters, and database just in case.

https://drive.google.com/drive/folders/1142YHaxB1RC6HMcfuy_MdEN8T-sSLDSl?usp=sharing

Thanks!

-Will

[Jimmy Eng]

Will,

XPRESS extracts precursor intensities from MS1 scans and your mzXML file contains only MS/MS scans.  So that's why XPRESS is failing because it's expecting to parse MS1 scans which aren't present in this file.

Jimmy

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[Will Comstock]

Good catch, I will try to redo the RAW conversion to include my MS1 scans. Thanks Jimmy!

-Will

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[Will Comstock]

Okay I got it to work. For the record, my solution was the following:

1. Rather than use the msConvert packaged with the TPP, I downloaded it separately from ProteoWizard.
2. I converted my RAW file with the following parameters:

• 
• (I am not sure if "SRM as spectra" is required, but "SIM as spectra" was key as all my MS1 scans were classified as SIM-MS scans)

      3. I then searched the resulting mzML file as normal.

Thanks again, Jimmy!

-Will

[Luis Mendoza]

Hi Will,

Glad you got this to work!

A potentially quicker solution is to enter those extra flags in the "additional options" box in Petunia:

You can find a full set of available options at the Proteowizard msconvert info page:

https://proteowizard.sourceforge.io/tools/msconvert.html

At some point we'll add more options to this page as well.

Cheers,

--Luis

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