Processing data with heavy labelled proteins and/or peptides

[SStoychev01]

Hi,

How would one process DIA data in DIA-NN from a digested lysate spiked with Heavy Lys and Arg 13C and 15N labeled protein standards?

Thanks,

Stoyan

[vdemichev]

Hi Stoyan,

Should be quite easy:

• Generate a predicted lib from FASTA
• Export to .parquet
• Open in R and replace each 'R' and 'K' with 'R(SILAC)' and 'K(SILAC)' just for spiked-in sequences
• analyse using commands as in scenario 2 step 2 here: https://github.com/vdemichev/DiaNN?tab=readme-ov-file#multiplexing-using-plexdia

Best, Vadim

[SStoychev01]

Thanks Vadim, will give this a go.

Best,

Stoyan

[JM-Bader]

I am also interested in searching DIA data for heavy-labeled peptides and then loading the data into Skyline, to build a subsequent targeting assay.

1) Is it possible to have only C-terminal Ks and Rs labelled (SILAC mass shift) ? I have a few miscleaved spike-in peptides that have an internal K or R. Manipulation of the parquet file is easy, but does DIA-NN allow both labeled and unlabeled K and R within a peptide? 2) Let's say my sample contains these heavy spike-in peptides only, no unlabelled background proteome. This is useful to build a library for targeting. I guess it should be possible to run this with fixed modifications directly in DIA-NN. How would one do that?

I tried this, which however crashed during the processing of the first raw file: --fixed-mod heavyK,8.014199,]^K,label --fixed-mod heavyR,10.008269,]^R,label --lib-fixed-mod heavyK --lib-fixed-mod heavyR --original-mods

3) .parquet files are generated from DIA-NN 1.9.1 and higher, correct? Can the modified .parquet library be used for a search by DIA-NN 1.8.1 (or any 1.8 version)? The reason is that Skyline appears not to properly load DIA-NN results from 1.9 versions.

thanks!