Random prep for Mass Spec
There were a few last items that needed to happen prior to injecting samples into Mass Spec. Here they are!
Make peptide histograms from past geoduck samples
- Log on to Proteomics Resource site; *need (credentials)
- Find geoduck peptide .xml files via the following path:
- emmats -> TPP -> geoduck -> transcriptome -> select any 10 files with the format “interact … pep.xml”
- There were more than 10 .xml peptide files, so I used a random number generator to select the following 10 files: 11, 24, 27, 29, 31, 33, 65, 67, 84, 87
- For each file, go to pick columns -> add mz ratio column -> update -> other actions -> export
- After exporting, use FileZilla to transfer 10 .xml files to local computer (need credentials)
- Combined data in 10 files into 1 file via excel (pretty straight forward).
- Used R Studio to generate a histogram (for all 10 samples combined) of m/z ratio:
- From this histogram Emma suggested that we go with a 450-850 m/z analysis window for my samples.
Add PRTC to samples
We must add a known amount of PRTC QC peptide mix to each sample, which provides a standard concentration of known peptides. Here’s how we did it:
- Ordered Peptide Retention Time Calibration Mixture ThermoFisher #88320. This is 50ul of 0.5pmol/ul
- Diluted PRTC to 200 fmol/ul
- Pipetted 20ul of PRTC into new tube
- Added 30ul acetonitrile, vortexed & centrifuged lightly
- Final volume is 50ul of a [200fmol/ul]
Prepare new sample tubes:
- Pipetted 3.125 acetonitrile into new, labeled sample tubes
- Added Pipetted 10ul of each sample into new tubes (each sample has protein concentration of 0.5ug/ul)
- Added 1.875ul of the 200fmol/ul PRTC
- Vortexed & centrifuged lightly
- Final volume is 15 ul of sample + PRTC + Acetonitrile. Should have 5ug of peptides total in each tube.
Pack Mass Spec columns:
- We watched/helped Emma pack the columns for the Mass Spec. This was a very precarious/labor intensive step. I will not go into the details here, but this step should be started at least a few days prior to starting on the Mass Spec, as the column can take several hours to pack, and one needs 2-3 columns.