Context: I am a student-researcher. I expressed GST-tagged protein (Pyruvate Kinase) in the yeast Saccharomyces cerevisiae on synthetic drop-out media. I then purified the GST-tagged protein, ran it in a polyacrylamide gel and sent the band containing the purified proteins for Mass Spectrometry. The proteins were subject to trypsin digestion and subsequently LC-MS/MS, and analysed using ProteinPilot software (SCIEX). I am quite unsure of the analysis parameters using the software as it is not up my alley.

Generally, is R-deamidation considered an artefact of MS? Deamidation usually occurs on asparagine or glutamine residues. However, I observed a large amount of peptides with arginine deamidated, and I am wondering whether arginine deamidation is biologically significant or simply an artefact of trypsin digestion / MS preparation. I also wondered if it could be the S. cerevisiae way of mimicking protein citrullination, a PTM observed in humans, since yeast has no known enzyme with arginine deiminase activity. However, I have my doubts about this, and and I would just like to clarify if anyone knows about the significance of R-deamidation for proteins.

Additionally, I am aware that oxidation may be an artefact of the GST protein purification process. Would there be any way to tell a functionally relevant oxidation modification from an artefact generated through purification without testing every single modification observed?

Thank you so much! I apologise in advance if I was unclear with anything.



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