laurahspencer
commented
7 years ago Thanks, Emma.
So, to confirm @yaaminiv and I will be doing DIA analysis on our samples. We see that this includes the extra optional digestion step:
"For DIA analyses: Add ApoA1 to each sample (dilute 1.1 µl 2200 ng/µl stock in 48.9 µl of 50mM NH4HCO3 to make 50 ng/µl) to make 1:300 ratio of ApoA1:total protein. For 100 µg total protein in a sample, add 333 ng of ApoA1 by adding 6.7 µl of 60 ng/µl ApoA1 solution."
Since we are working with 30ug instead of 100ug, we will add 2.2ul of the prepared 60 ng/µl ApoA1 solution. Let us know if we've misinterpreted anything.
emmats
yaaminiv
sr320
Ellior2
seanb80
Here are general guidelines for analysis of data dependent acquisition acquired mass spectra (or, shotgun proteomics). @Ellior2 will be following these steps, Laura and Yaamini will be doing something slightly different and should see notes on DIA analysis.
Comet installed and an appropriate comet.params file
Your raw experimental files (excluding blanks and QCs) in one directory
a protein sequence fasta file that you expect is a good match to your acquired data
I usually soft link my files to a specific search directory (link -s). The comet.params file must be in the same directory. Make sure your parameters reflect the type of data you have and that the database path is correct. More details can be found on the UWPR website. It is good practice to include a decoy search. If searching all files in the directory, you can simply type: runCometQ *.raw
TPP installed
pep.xml search results from Comet I usually run TPP in the same directory as my Comet searches. You need to run it on each file individually, but each analysis is relatively fast. I use a p-value cut-off for my peptide probability of 0.9, but this can be changed depending on what you want. For each file, run: xinteract -p0.9 -OAp -dDECOY_ -N[file name without pep.xml] file name.pep.xml
ABACUS installed
interact -....pep.xml files from TPP
An Abacus parameters file Details can be found on the UWPR tools page. Once ABACUS runs, you will have a .tsv file with all your files joined together and corresponding spectral counts and normalized spectral abundance factor (NSAF), which will be your proxy for protein abundance.