Hi to all, I'd like to know if it would be possible that some amino acid modifications occur chemically during protein sample preparation or during the mass spectra acquisition. For example is it possible that at certain condition some amino acid residues are decarboxylated? What could be the possible modifications during sample preparation?
Thanks
Dear Antonio,
yes unfortunately proteins can be modified during experimental preparation. There are several examples of protein modification that can occur at the different steps of protein analysis (precipitation, separation, digestion, ...). Some of those examples are oxidation of methionine (Met), tryptophan (Trp) to kynurenine derivatives, and depending on the experimental conditions used in precipitation, separation and digestion you may also find oxidation in aromatic amino acids such as His, Phe, Tyr, others. Not so long ago, some researchers focused on this problem, pls follow the link (http://www.ncbi.nlm.nih.gov/pubmed/20219394). Although protein modification cannot be prevented it can be minimised and use the results obtained to our advantage.
Hope this helps,
Ana
Dear Ana, i think there is a problem with the link you posted. could you please give me the dpi of the article, or the authors, the title?
thanks
Take care of the treatment conditions to evit changes in lateral chains of amino acid residues. Do not make suddenly pH increasing or decreasing, take care also of buffers' composition and pH. Hydratation, mixing and solvatation are very important if your protein was previously dried. Drying process also can change the protein conformation and properties.
I would like to add that you should take into account what kind of methods you use during your sample preparation. Some modifications are method-associated (e.g. carbamyl after heating in high urea before EP). However, during the search you can select a list of the most probable modifications (depending on your sample handling). In some programs, e.g. in Paragon algorythm (ABSciex ProteinPilot protein ID software), you can select the method of sample preparation you used itself and the program would use a list of appropriate modification automatically. I have not heard of any really problematic modifications during the ionization step, but when doing MS/MS you would always have a set of different forms of ions (plus water, hydrogen and so on).
During sample preparation, some amino acid modifications might occur. Probably the most frequent modification is oxidation of methionines.
If you work with urea, you might get carbamylation i.e. of lysines (see Sergey's comment). Carbamylation is more prominent if you heat up the sample in the presence of urea. Some protocolls suggest to do enzymatic digestion at room temperature to reduce this unwanted modification.
Deamidation of asparagine is also frequently observed. It is an post-translational modification, but it can also happen during sample prep:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3205879/
Also pyro-Glu or pyro-Gln can be found. This modification occurs especially at when E or Q are at the N-term of the peptide. It occurs quite frequently if you use AspN for digestion.
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