Using PepQuerry for identification of protein-protein interaction

[anuC]

Hi,

First of all, thank you for such wonderful tool. I'm working with proteins translated from new ORFs and I predicted that the new protein interact with another protein, say for e.g. protein X. Now, I would like to validate this prediction with analysis of available AP-MS datasets for the protein X (bait). Will it be possible to use PepQuery here, to look for whether the new protein present in the interactome of the protein X (using a related approach of 'validating known peptide identification' from PepQuery2 article - Nat. Commu. 2023, 14, 2213).? If so, as a beginner with no prior experience in MS data handling, what are the different steps I should follow after downloading the corresponding thermo raw data?

Any help would be highly appreciated

Thanks in advance
Anu

[wenbostar]

The short answer is Yes. Let's move the discussion to email. What is your email?

[anuC]

Thank you for the prompt reply. My email ID is [email protected]. Any help would be highly appreciated.

[anuC]

Hi Bo,

Follow up with my analysis for protein-protein interaction, I have downloaded the bait protein spectra from Bioplex in RAW format. Later, the raw files were converted to mgf format with msconvert GUI using the command -
C:\Users\msconvert.exe" --mgf --zlib --filter "peakPicking vendor msLevel=1-" --filter "titleMaker <RunId>.<ScanNumber>.<ScanNumber>.<ChargeState> File:"""^<SourcePath^>""", NativeID:"""^<Id^>"""" "b8445.raw" "j9529.raw"

mgf files were later analysed with PepQuery, with both 'known protein validation' and 'novel protein validation' approaches, as shown below

For known protein approach, I have added the protein of interest in the reference database in fasta format - java -Xmx40G -jar pepquery-2.0.2/pepquery-2.0.2.jar -db reference_db.fasta -ms combined.mgf -hc -s 2 -m 1 -o PepQ-out/ -i ch_450 -t protein -fast -tol 50 -itol 0.05

For novel protein validation - java -Xmx40G -jar pepquery-2.0.2/pepquery-2.0.2.jar -ms combined.mgf -db RefSeq_HomoSap.fasta -hc -s 1 -m 1 -o Pep_450_novel -i MARAGNPAPSRWTKTTAAPTRRTAPRREPSTGWWTWRGKPTSCRPRRAWTMEAVFKASSS -t protein -fast -tol 50 -itol 0.05

Am I doing it correctly?. It would be great if you could let me know your comments here.

Many thanks in advance
Anu