Generation of spectral library using Prosit

Generation of spectral library using Prosit benoit fatou  2020-03-03

Hello Skyline Team,

I am trying to use Prosit to generate spectral libraries and I realized that Prosit does not recognize "propionamide" as a fixed modification on the cysteine residues even when I set this modification on Skyline.
Is there a way to modify the modifications into Prosit?

Thanks for your help,

Brendan MacLean responded:  2020-03-03

Do you have a hack that works for you with the Prosit website? If you can come up with one and you can explain it clearly, then we will consider making it possible in Skyline. Right now we only allow the modifications we know Prosit supports. And we don't have a way of asking Prosit for intensities using one set of modifications and pretending the spectrum intensities belong to another set of modifications if that is what you are asking for.

Do you have strong support for the idea that "propionamide" would not impact peptide fragmentation (and retention time) when compared with some other cysteine treatment?

Thanks for your feedback and for trying out the latest support for Prosit spectrum prediction.


benoit fatou responded:  2020-03-03

Unfortunately I don't have a hack that works and I don't know at 100% that the carbamidomethylation and the propionamide modification would give rise to the exact same MS/MS spectrum.
Sorry for the lack of explanation.


Brendan MacLean responded:  2020-03-03

I guess I would recommend doing that experiment first. Predict a bunch of spectra with Carbamidomethyl (C) and then compare them against what you get with propionamide. If the match is good enough, maybe you can get the Prosit team to support propionamide.

benoit fatou responded:  2020-03-04

Hi Brendan,

I just did a quick PubMed search and I found a paper (see below) where they compared the iodoacetamide and the acrylamide and they shown a better efficiency using acrylamide in terms of peptide coverage. I know this people was published almost 20 years but it might be a good starting point. in the same time, I will look at my own data and provide you additional information.
Thanks for your help,

Proteomics. 2002 Dec;2(12):1672-81.
In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
Mineki R1, Taka H, Fujimura T, Kikkawa M, Shindo N, Murayama K.
Author information

Cysteinyl residues in proteins were alkylated with acrylamide during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to yield a thioether derivative, cys-S-beta-propionamide (PAM cys). The process was termed in situ alkylation with acrylamide. Using this method, the recovery of PAM-cys peptides from bovine serum albumin (BSA) was 88.6% at 10 picomol in one-dimensional (1-D) SDS-PAGE and 97.1% at 50 picomol in two-dimensional (2-D) SDS-PAGE. The coverage of tryptic peptide of BSA in 1-D and 2-D SDS-PAGE was 83.7% and 81.1%, respectively. The advantages of in situ alkylation with acrylamide were the following: (i) cysteinyl peptides were effectively derived in a single PAM cys and then proteins were precisely identified using databases; (ii) marked reduction of salts compared with post alkylation, e.g., using carboxymethylamide (CAM), resulting in higher signal intensity and wider coverage of cysteinyl peptides from PAM cys, compared with those of CAM derivatives, in mass spectrometry peptide mapping; and (iii) shorter duration by excluding the processes of post alkylation and desalting before peptide mapping.

Brendan MacLean responded:  2020-03-04

The question is not whether more spectra get identified, but whether the different treatment preserves the same relative fragment ion intensities and elution times. i.e. can you use predictions based on one treatment for the other?

benoit fatou responded:  2020-03-10

Dear Skyline Team,

Please find attached some results showing the similarities of iodoacetamide and acrylamide as a alkylation reagent in terms of retention time and MS/MS spectra.
Let me know if you need additional information.

Brendan MacLean responded:  2020-03-10

Well, it looks like this is not actually very hard to achieve, based on how I read the code in Skyline:

   private static readonly HashSet<PrositAA> PrositAAs = new HashSet<PrositAA>()
        new PrositAA('A', 1), new PrositAA('C', 2), new PrositAA('D', 3),
        new PrositAA('E', 4), new PrositAA('F', 5), new PrositAA('G', 6),
        new PrositAA('H', 7), new PrositAA('I', 8), new PrositAA('K', 9),
        new PrositAA('L', 10), new PrositAA('M', 11), new PrositAA('N', 12),
        new PrositAA('P', 13), new PrositAA('Q', 14), new PrositAA('R', 15),
        new PrositAA('S', 16), new PrositAA('T', 17), new PrositAA('V', 18),
        new PrositAA('W', 19), new PrositAA('Y', 20),

        // Mods
        new PrositAA('C', 2, UniMod.DictStructuralModNames[@"Carbamidomethyl (C)"]),
        new PrositAA('M', 21, UniMod.DictStructuralModNames[@"Oxidation (M)"])

I other words, Prosit has a special AA index (21) for an oxidized M (versus 11 for an unoxidized M), but C and Carbamidomethyl (C) just get treated as synonyms (index 2). So, I should be able to add the synonym you are requesting with a single line:

       new PrositAA('C', 2, UniMod.DictStructuralModNames[@"Propionamide (C)"]),

Would you agree?

In the end, Skyline sends a vector of indexes to Prosit and gets back a matrix of corresponding fragment intensities. It is already up to Skyline to assign those intensities to m/z values. So, it should deal seamlessly with the change in mass between the two modifications.

benoit fatou responded:  2020-03-10

Thank you very much Brendan for your help !
I 100% agree with you.
Let me know when the new update will come.

Thanks again,