Today I got some more input from Emma regarding the reports to export from Skyline to analyze. I exported a new MS Stats report (the built-in one from Skyline) and also made the bioreplicates in the file accurate (details in post). Additionally, I exported a new report that includes information that should help us get to the point of comparing 23C and 29C proteins, and identifying the proteins expressed in total.
New reports
New MS Stats report
New protein list
The new protein list integrates the transitions for the each peptide. However, we are still trying to figure out how to integrate the peptides for each protein (GitHub #666).
I also spoke with Yaamini, and she told me that we should only look at proteins that have 3 or more peptides (ones that have 2 or 1 should be discarded). The ones that have more than 3 peptides, the three most abundant peptides should be used.
Re-do MS Stats with new report
Products from new MS Stats:
- protein comparison results table
- Comparison Plot
- Profile Plot
- Profile Plot with Summarization
- QC Plot
- Volcano Plot
Thoughts on new MS Stats
I haven’t delved too much into the plots, except for the Volcano Plot.
It is very different from the one I did with the earlier MS Stats report (the one I created by hand).
The one from before had the bioreplicates wrong. I had:
| Temp trtmnt | sample day | sample | bioreplicate |
|---|---|---|---|
| 23C | 9/11/15 | 1 | 1 |
| 29C | 9/11/15 | 2 | 2 |
| 23C | 9/14/15 | 13 | 1 |
| 29C | 9/14/15 | 14 | 2 |
And it showed that there was 1 significantly downregulated (old volcano plot)
Today, Steven said that bioreplicates should actually be within treatment. So I fixed the bioreplicates for today’s MS Stats report and MS Stats analysis in R. The bioreplicates are now:
| Temp trtmnt | sample day | sample | bioreplicate |
|---|---|---|---|
| 23C | 9/11/15 | 1 | 1 |
| 29C | 9/11/15 | 2 | 1 |
| 23C | 9/14/15 | 13 | 2 |
| 29C | 9/14/15 | 14 | 2 |
Maybe that’s why the new Volcano plot has no significant proteins and the last plot did.
Next steps:
- Compare 23C with 29C using the new report: 0403-DIA-Cgseed.csv
I also spoke with Yaamini, and she told me that we should only look at proteins that have 3 or more peptides (ones that have 2 or 1 should be discarded). The ones that have more than 3 peptides, the three most abundant peptides should be used.
-
Get protein lists: total proteins, and (if temp trtmnts are different) proteins for each temp trtmnt
-
Clean up repo: readme files; only include important stuff
-
Finish paper by April 17th