Surprisingly there is little concrete info on this. If you biotinylate a protein/peptide (in this case with Sulfo-NHS-LC-Biotin) through a primary amine at the N-termini or lysine side chain, can that amine be protonated for electrospray MS? In other words, if you biotinylate the N-termini of a peptide that has a C-terminal arginine, will it be singly or doubly charged?
As a short answer the reaction of NHS-esters with peptides (such as Sulfo-NHS-LC-Biotin) will lead to the formation of a new amide bond. Since the proton affinity (equivalent to the basicity) of amides is lower than that of amines, the biotin derivatisaton is likely to reduce by one the number of basic sites in a given peptide.
The major problem in answering your question is that the charge state of a peptide in ESI depends also on other factors than the number of basic sites. So I would say that empirically (and according to my personal experience on peptide derivatisation using NHS activated esters) you are likely to observed a reduction by one in the peptide global charge states.
As a short answer the reaction of NHS-esters with peptides (such as Sulfo-NHS-LC-Biotin) will lead to the formation of a new amide bond. Since the proton affinity (equivalent to the basicity) of amides is lower than that of amines, the biotin derivatisaton is likely to reduce by one the number of basic sites in a given peptide.
The major problem in answering your question is that the charge state of a peptide in ESI depends also on other factors than the number of basic sites. So I would say that empirically (and according to my personal experience on peptide derivatisation using NHS activated esters) you are likely to observed a reduction by one in the peptide global charge states.
Thanks Guillaume. I suspected as much. But I think I might do the experiment on a model peptide to make sure. On another note, some of the MSMS spectra of peptides we suspect of being biotinylated (in a global untargeted labelling experiment) have an intense fragment ion at 310Da that we simply can't assign. Anyone have any idea what this could be?
It is very difficult to predict the charge state of a protein as Guillaume already pointed out. The number of basic/acidic sites is not always a key factor influencing the protonation degree of your protein. The reaction with the NHS activated esters might cause a perturbation in the structure of the protein, burying or revealing additional protonation sites regardles of the nature of your substrate. This effect is probably absent/negligable in the case of small peptides. Moreover, I think that the NHS activated ester might also react with additional primary amines (e.g. Lys side chain) in your protein/peptide, complicating things even further.
The 310Da ion is actually the biotin (226Da) plus a lysine immonium ion (84), as revealed by a Protein Science paper (2009, 18:314-328).
My logic is that by biotinylating on the protein level, we block all lysine and the N-term and thus trypsin won't cut at lysine, making it an Arg-C digest. Thus, internal peptides will still be 2+ because of the new peptide N-term and the C-term Arg. But the original N-term peptide will be 1+ because there is only the C-term Arg to be protonated (unless you are lucky enough to have a His in the peptide). I just need to do the experiment.
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