Skyline works with our demultiplexed files!

Emma sent over a test .blib made from my demultiplexed and converted oyster 1 mzML file. Using the new Windows machine, I ran this file through Skyline Daily. I followed settings Emma suggested as well as the protocol from my previous Skyline test.

Peptide digestion settings

Adding my background proteome.

My background proteome FASTA file can be found here.

Modifying my peptide digestion settings.

For digestion enzyme, I used Trypsin.

Modification and Quantification.

Last time I used Skyline, I did not touch these sections.

Adding FASTA file

I imported the FASTA file like I did previously, but it did not work.

Error message from my attempt to import my FASTA file:

Transition settings

I decided to move on and change my transition settings before I figured out how to add my protein sequences.

Adding protein sequences

I opened up my FASTA file in Notepad and literally copied and pasted my sequences into the lefthand bar. It worked!

Importing my raw files as “Results”

Added oyster 1 to ensure it worked. It did!

I then imported the rest of my files.

Now that my Skyline file is set up, all I need to do is replace the .blib with one from all of my mzML files and start picking targets for SRM.