error in converting Spectronaut 19 file to MSstats PTMFormat

[Wing Man Ko]

Hi all,

I have encountered an issue when converting Spectronaut 19 file to MSstats PTMFormat for my phosphoproteomics analysis. 

Please see the codes below and the attached screenshot. 

names(spectronaut_input)

unique(spectronaut_input$R.Label)

spectronaut_annotation = data.frame(
    Run = unique(spectronaut_input$R.Label),
    Condition = c("CNT", "LIM", "LIM", "CNT", "LIM", "CNT", "CNT", "LIM"),
    BioReplicate = c(1, 1, 2, 2, 3, 3, 4, 4)
)

spectronaut_annotation

msstats_input = SpectronauttoMSstatsPTMFormat(spectronaut_input,
                  annotation = spectronaut_annotation,
                  fasta_path = "01_raw_data/uniprotkb_gallus_gallus_isoform and unreviewed_fa_2023_10_13.fasta",
                  use_unmod_peptides=TRUE,
                  mod_id = "\\[Phospho \\(STY\\)\\]",
                  fasta_protein_name = "uniprot_iso"
                  )

I will be very grateful for any help. 

Best, 

Winky 

[Anthony Wu]

Hi,

I saw a similar issue recently.  Did Spectronaut recently update the columns of its output file?  For example, in the past, it had a column called F.FrgLossType which was used for additional filtering.  I believe any Spectronaut update can cause issues from our side, which means we'll need to update our converters.

To assist further, would you be able to provide a sample of your data to help enable us to debug further and make updates to the converter?

Thanks,

Tony

[Wing Man Ko]

Hi Tony,

A sample of the data and the annotation can be found in the link below. Thank you so much!!

https://drive.google.com/drive/folders/1U068Rw-2rhAljGlk3ns1-4m8zAFPBbTE?usp=share_link

Best,

Winky 

[Anthony Wu]

Hi Winky,

By any chance, did you choose to include fragment ion level information in your report?  MSstats relies on Spectronaut's fragment headers to perform differential abundance analysis, but your report does not have any fragment headers.  I'm reading the user manual for Spectronaut 19 and it looks like fragment headers can still be included in the report.  

Thanks,

Tony

[Wing Man Ko]

Hi Tony, 

It works after adding the fragment headers. Thank you very much for your reminder!

Best regards,

Winky