Making libraries for synthetic peptides and packaging them into single file in skyline

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Making libraries for synthetic peptides and packaging them into single file in skyline SRS  2020-07-07
 

Hi There,

I have been trying to make a spectra library of about few thousands synthetic peptides from a DDA run. I was able to import the data using "mziden" and "mzxml" files from the search and mass spectrometry data, respectively. But I have two questions here, 1) how can I export all the spectra libraries into a single file in skyline?

  1. How could I do the peak picking for over thousand peptides, as they are so many, it is not possible for me to open them to make sure the RT is aligned.

Do I need to make sure the alignment of the RT is correct then package them?

I can send you the skyline file if that help,

Thank you, looking forward to hearing from you.

Regards,
SRS

 
 
Brendan MacLean responded:  2020-07-07

Sure. Go ahead and post the Skyline document (use File > Share to create a .sky.zip file) to:

https://skyline.ms/files.url

It would be helpful to see what your library looks like. It sounds like you are saying you did one DDA run with a sample mix containing thousands of synthetic peptides. Or was it multiple DDA runs? If multiple runs, did they have any peptides in common?

Thanks for posting to the Skyline support board.

--Brendan

 
SRS responded:  2020-07-07

Hi Brendan,
Thank you very much for the quick response.Yes, this is a mixture of thousands of synthetic peptides in the form of XXXXXXXWLR where X is a random combination of seven amino acids.We ran this mixture in one DDA run ( I included in the zip file)
I wonder if there is a way that I can export all the spectra libraries of the good identified peptides (about 500 of them) between these thousands into a single file in skyline? How could I do the peak picking for these over thousand peptides, it is not possible for me to open them to make sure the RT is aligned. I am worried about it. We ran these using a fusion orbitrap lumos.
Do you think that I need to make sure the alignment of the RT is correct then package them?I tries to upload the zip file to the web-page you gave me. It is called "XXXXXXXWLR"

Thank you so much for the incredible help,

SRS

 
SRS responded:  2020-07-10

Hi Brendan,

Just wanted to follow up with you, if you got a chance to check the file. Really appreciate it.

Regards,
SRS

 
Tobi responded:  2020-07-13

Hi SRS,

for DDA we have good experience with loading the data with transition settings DDA centroided X ppm, triggered acquisition checked for good peak picking. (Eventually combined with +- X minutes around MSMS IDs).

You can further filter for RT alignment, idotp and dotp via document grid - action - delete precursor as you deem it reasonable.

You can convert your measurement data into a library with File - Export - Spectral Library.

Best,
tobi