Here are the parameters I set (only listing what I changed from default) and some questions we may want to investigate: This is for clarified cell extracts that were used to determine %H-incorporation and %H-mixing
(1) No “experiments” set - each sample treated as its own experiment —> we had a question whether e.g. grouping the heavy-label-only experiment for determining incorporation) with the heavy-light mix experiment might be advantageous for "matching to and from" and/or "matching between runs."
(2) I checked the “Re-quantify” option in group-specific parameters: Misc tab (this SHOULD allow large H/L ratios to be detected when one label is in/near the baseline. —> how does the “match type” option affect RAW files that are specified as different experiments? Do these need to be grouped? How does this work?
(3) I unchecked “Second peptides” option in Global parameters: Advanced ID tab (one peptide per MS/MS should be OK for assessing labeling and mixing and reduce run time) —> by default “match between runs” is unchecked. How does this option interact with "re-quantify" and "match to and from” above?
(4) Folder location tab —> I have set a static Temp folder and Andromeda folder. Keep these constant for now (in the future I will set up an SSD for temp files). However you should always point the combined folder to be within the “analysis” directory of the folder you are working from (with MQ prefix). As I said, follow the directory structure I have set up.
(5) for protein quantification I have set 3 min ration counts
(6) I added Carbamidomethyl (C) to the list of modified peptides that can be quantified. List has Oxidation (M), Acetylation (N-term), and Carbamidomethyl (C). I actuall forgot to add Carbamidomethyl so I just added it and re-started the run :( - note, taken together these are all the variable and fixed mods specified in the params. We should find out if “fixed” mods have to be listed here, since, ALL peptides with C are presumed to be modified, seems like fixed mods would automatically be included here without added them (especially since Oxidation (M), Acetylation (N-term) are added by default whereas Carbamidomethyl (C) is not, despite being a default fixed mod). So… probably I terminated my run for no reason, but, whatever. Please look into the significance of this option. Also, by N-term acetylation, does it mean N-term as specified in the FASTA file provided? I guess the role here is… the MS1 intensity of the modified and unmodified peptides are pooled? What’s actually happening here?