Hi all,
I was analysing a phospho-proteomics sample (tryptic digest from a human cell line) and, as the number of phosphosites I could identify was surprisingly low, I ran MaxQuant with dependent peptide search to see if anything unexpected would come up. Indeed, about 400 peptides were interpreted as formylated (compared to about 2000 regular peptide IDs). As many of them seem at the peptide N-terminus, they look like a sample prep. artefact.
I was wondering if any of you has seen this before and if so what it is caused by?
Thank you,
Michael
Hi Markus,
thanks.
We used a quite standard in-solution digest procedure. The only non standard thing was that we dried the samples after digestion and acidification and before clean-up, because the collaborators that harvested the cells and shipped the peptides didn`t have the equipment:
-harvest attached human cells in buffer with 8M urea
-reduce alkylate: TCEP + iodoacetamide
-digest: LysC + trypsin
-spin out debris, acidify to 0.2% FA, speedVac, ship samples
-SepPak clean-up; lyophilize
-phospho-peptide enrich with magnetic beads: binding buffer with 1M glycolic acid, 5% TFA, 80% ACN
elute in 1% ammonium hydroxide
acidify with TFA ~2%
-clean with C18 spin cartridges; solutions contain 0.1% FA
-lyophilize
Let me know if I should give more details somewhere.
Best,
Michael
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