Issues running Spectronaut data from MSstatsPTM LabelFree Workflow Vignette

[joreynajr]

Hi,

I cloned this repo and updated the MSstatsPTM LabelFree Workflow Vignette so that this:

msstats_input = SpectronauttoMSstatsPTMFormat(spectronaut_input, 
                  annotation=spectronaut_annotation, 
                  fasta_path=system.file("extdata", "spectronaut_fasta.fasta", 
                                         package="MSstatsPTM"),
                  use_unmod_peptides=TRUE,
                  mod_id = "\\[Phospho \\(STY\\)\\]",
                  fasta_protein_name = "uniprot_iso"
                  )

Becomes the input of:

MSstatsPTM.summary = dataSummarizationPTM(msstats_input , verbose = FALSE, 
                                          use_log_file = FALSE, append = FALSE)

However, when I do this, both of the following fail:

dataProcessPlotsPTM(MSstatsPTM.summary,
                    type = 'QCPLOT',
                    which.PTM = "allonly",
                    address = FALSE)

# I don't understand this error
Error in if (unique(datafeature$LABEL) == "L") { : 
  argument is of length zero
Error during wrapup: not that many frames on the stack
Error: no more error handlers available (recursive errors?); invoking 'abort' restart

and

dataProcessPlotsPTM(MSstatsPTM.summary,
                    type = 'ProfilePlot',
                    which.Protein = "Q9Y6C9",
                    address = FALSE)

# this should exist, I checked the summary object for it 
Error in .profile.lf(data.table.list, type, ylimUp, ylimDown, x.axis.size, :
Please check protein name. Dataset does not
have this protein. - P36578 

I appreciate your help as I'm trying to run MSstatsPTM for data generated by Spectronaut.

Note: all other functions work.

Joaquin

[tonywu1999]

@joreynajr

Could you update to R 4.6.0 and try installing MSstatsPTM with the following code:

if (!require("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

BiocManager::install("MSstatsPTM")

I installed from Bioconductor and wasn't able to reproduce the issue from my side.

[joreynajr]

Thanks for that @tonywu1999 , I will give it a try and keep you posted.

[joreynajr]

Hi @tonywu1999,

I installed R 4.6.0 plus MSstatsPTM. The lines that were previously failing now work but now I'm having a new issue. This here:

MSstatsPTM.model = groupComparisonPTM(MSstatsPTM.summary, 
                                      ptm_label_type = "LF",
                                      protein_label_type = "LF",
                                      contrast.matrix = "pairwise",
                                      use_log_file = FALSE, append = FALSE,
                                      verbose = FALSE)
head(MSstatsPTM.model$PTM.Model)
head(MSstatsPTM.model$PROTEIN.Model)
head(MSstatsPTM.model$ADJUSTED.Model)

Fails with the following error message:

Error in MSstatsContrastMatrix.matrix(contrast.matrix, groups) :
Assertion on 'colnames of contrast matrix' failed: Must be a permutation of set {'H0_Y100','H100_Y100','H100_Y150','H100_Y200','H100_Y25','H100_Y50'}, but has extra elements {'H100_Y0'}.

Do you know what this could be?

Many thanks,

Joaquin

[tonywu1999]

My guess is that the sample dataset has H100_Y0 as a factor in the GROUP column but none of the rows are listed with that condition (maybe due to earlier filtering).

Could you try to reset the factors of the GROUP column of the summarized data. Like below:

MSstatsPTM.summary$PTM$ProteinLevelData$GROUP <- factor(MSstatsPTM.summary$PTM$ProteinLevelData$GROUP, levels = c('H0_Y100','H100_Y100','H100_Y150','H100_Y200','H100_Y25','H100_Y50'))
MSstatsPTM.summary$PROTEIN$ProteinLevelData$GROUP <- factor(MSstatsPTM.summary$PTM$ProteinLevelData$GROUP, levels = c('H0_Y100','H100_Y100','H100_Y150','H100_Y200','H100_Y25','H100_Y50'))

[joreynajr]

Thanks! I'll give that a try!