hi I intend to expose my cells to mechanical stretching while in SILAC media.
Im a pretty inexperienced researcher being an undergrad but what kind of downstream mass Spec analysis can i perform and which should i use?
I have heard about looking at differences in phosphorylated proteins as well as changes in protein levels after cell stretching. Which approach should i use and for each approach is there a difference in subsequent steps?
Go to the web site: Uniprot
Do a phosphopeptide specific cleanup. Do an in silico trypsin digestion of the main proteins you are looking for. Pick cleavage peptides from loop regions without W and M residues with your labeled amino acid on it. Use these as targets for multiple reaction monitoring. You can create your acquisition method with Skyline.
Good luck.
Read before you do anything, e.g. "Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC): Methods and Protocols ", ed. B. Warscheid. Successful SILAC analysis requries many tweaks from cultivation to MS analysis to data processing. E.g. can the cells your are using be cultivated as 'heavy', i.e. K/R auxotrophic? Do you have a high resolution proteomics MS setup available, e.g. nanoLC-Orbitrap of any kind? SW is available for free - MaxQuant and Perseus - however need to be learned.
Second: look at the timescale of your experiment. Depending on the duration of your mechanical stretching, which biological effects might actually make sense? Changing protein expression takes a while, for example; phosphopeptide signaling may take place on the timescale of seconds to minutes instead. Have the effects of similar stresses (shear stress? pressure?) been desccribed on the proteomic level?
Discuss all this and more with your supervisor or other experienced biochemists at your location. You are basically asking about designing a full-blown proteomics experiment from scratch, and this is no task for an undergrad. If you supervisor or the people around you should not be able to at least guide you: run ;)
Good luck!
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