Which tutorial most nearly matched your experiment?

Best regards,

Brian Pratt

Hi Brian,

This is the link https://skyline.ms/_webdav/home/software/Skyline/%40files/tutorials/MethodEdit-3_7.pdf where i referred. I followed the steps for Yeast protein mentioned in the document.

But for example if my experiment is on **Infliximab**, I need to **download files from the online available libraries** i.e Peptide Atlas, GPM, NIST. But am not sure which files to be downloaded and also the file formats supported by Skyline for my experiment on 'Infliximab' Light Chain.

Kindly guide.

Thanks,
Shobha

Hi Shoba,

That's not enough information to yield any advice, sorry. Can you tell us in detail what your experiment has entailed so far? Especially what kind of mass spec analysis you've done and what your software pipeline consists of, and what files it has used and/or produced?

Best regards,

Brian

Hi Brian,

As I mentioned in my earlier emails that I am new to Skyline application and going through the tutorials and webinars to get more information.

I need to explore Infliximab as a case study in buffer, rat, and human matrices
- Develop and optimize MRM methods for the surrogate peptide approach between MassLynx and Skyline (method optimization for quantification of a digested protein)

**Workflow:**

Step 1 (Skyline)
Skyline initial process of peptide to create mrm transitions
Export to create acquisition method 1
Step 2 (MassLynx)
Acquire raw 1 data using method 1
Step 3 (Skyline)
Process raw 1 data to find RT of MRM transitions
Export method 2 to optimise CE
Step 4 (MassLynx)
Acquire raw 2 with method 2
Step 5 (Skyline)
Process raw 2 to find best CE
Export method 3
Step 6 (MassLynx)
Acquire raw 3 data using method 3 (Final acquisition method)

So to begin with the Skyline, I need to first have the spectral library setup (Peptide Setting). Then insert the protein in the Target for my experiement on Infliximab. Hence I need to **download files from the online available libraries** i.e Peptide Atlas, GPM, NIST. But am not sure which file or files to be downloaded for my experiment on 'Infliximab' Light Chain.

Could you please suggest.

Thanks,
Shobha

In continuation with my above mail, I forgot to mention that I need a Protein sequence or peptide sequence file too for **Infliximab LC**.

Thanks,
Shobha

We definitely can't help provide you with the antibody sequence. Also because Infliximab is a chimeric mouse/human antibody therapeutic you would probably need to target the mouse portions of the sequence if you are monitoring it in a human sample matrix. Is the sequence of the antibody publically available? With that and a trypsin digest of the antibody you can optimize a targeted assay as we have described previously for recombinant proteins.
https://www.ncbi.nlm.nih.gov/pubmed/22056677
https://www.ncbi.nlm.nih.gov/pubmed/22577014
https://www.ncbi.nlm.nih.gov/pubmed/26867746

Cheers,
Mike

Thanks for your reply , Mike!

But I still have a query.

Without knowing about the list of proteins or peptides in the Target, how can I proceed.

In the tutorials, Yeast was the Target. Therefore all the library files concerned to Yeast were available from GPM, Atlas etc. The FASTA files were also provided. Even the list of protein and peptide list were provided too.

But in case of Infliximab, how should I proceed.

Kindly reply.

Thanks,
Shobha

If you don't know your protein sequence you can't make an assay to the protein. At least not using the strategies we have developed. The issue you have is that it is not encoded by either the human or mouse genome. Infliximab is a man made drug that is a chimera of the mouse antibody that was humanized. You need to find out the protein sequence before you can start.The sequence of a chimeric antibody drug will not be any of the fasta files because it isn't encoded by the genome... it is a drug. Is the sequence publically known? Maybe it is reported as part of the paper or patent? If not then your only hope is to get the sequence from the drug manufacturer or derive it manually (not a strategy we support).
Best of luck.
Mike

Thanks Mike for your detailed explanation.

Please find attached the screenshot. The highlighted one in Yellow is the sequence right. So this is what i need. But i don;t know the source.

Thanks,
Shobha

Hi Skyline Team,

Could you please reply to my earlier email.

Thanks,
Shobha

We obviously can't answer that question. It is up to you to be the expert in your protein of interest. With the sequence and a digest of your drug, you can test if it is working using the methods we suggested.

With the tutorials on this website and the papers mentioned above you have how we would approach the problem. It is up to you to figure out if it works for you.

Best of luck,
Mike