Assaying for Vitellogenin abundance in Geoduck

[shellywanamaker]

@emmats we're trying to see if we might be able to use a commercial vitellogenin ELISA kit with Geoduck hemolymph samples. I'm wondering where I can find the protein/peptide sequence(s) for the Vitellogenin (cds.comp144315_c0_seqI|m50928) you detected in your proteomics paper, so we can share it with Diapharma to see if it would be compatible with their antibody. And I'm also wondering what your thoughts are on other potential assays for this. I know you mentioned SRM in lab meeting, and did it in your paper, but how straightforward and economical would SRM be for this application? THX!

[emmats]

The protein sequence would be in the protein fasta file, that I believe @sr320 has, but I have copied the protein sequence below. Do you want the peptides that I actually used in the SRM assay?

I did ask Charlotte Corporeau if she knew of any assays. She said she would ask around and I have not heard back. I'm not well informed on other types of protein assays. My answer would be ELISA or Western Blot.

Since we have already done SRM in geoduck hemolymph and know we can detect it, you wouldn't necessarily have to do intensive mass spec to develop the assay. I think it would be worth measuring as many Vg peptide transitions as possible in a few samples and seeing which ones look the best and change abundance as expected with advancement of reproductive stage.

The mass spec for doing SRM costs about $90-155 per day (we have two prices in the spreadsheet and I'm not sure why...). It takes about 1 hour to run 1 sample.

Let me know if you want to know more - via github or in person. I'm only 2 blocks away.

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[shellywanamaker]

Thanks so much @emmats . It would be great to catch up more about the SRM stuff in person, especially since you're so close. Maybe sometime Friday or next week?

[emmats]

I should be around Friday, although I may have something in the morning. Do you want to meet around 11 or 12? I can come over to FTR.

[shellywanamaker]

Sure, either time works. I'm in 234.

[emmats]

I'll aim for 11:00. See you then.

[shellywanamaker]

great thanks @emmats

[kubu4]

@emmats - Can you please post link to FastA file? I didn't see any reference to a FastA file in the paper...

[emmats]

Again, @sr320 made the file so he probably has a link somewhere although I looked in cnidarian and couldn't find it. You'll note in the methods that a publicly available transcriptome was analyzed with Transdecoder to get the proteome for analyses. Here is the file: https://drive.google.com/open?id=1FfyuLyJ3Hfp9PS4qzed8_Sol3n9EfoXW

[kubu4]

Thanks for link to file. That's all I wanted (FastA or not).

[kubu4]

That link you provided is a/the FastA file!

[emmats]

Yes, I know. I just knew that Steven already had it somewhere on the Roberts Lab cluster and if he had been able to provide the link it would have been simpler. Of course I had it. I did the analyses.