Overalkylation of a protein digest with iodoacetamide

Cystine linkages in proteins are often opened with reducing agents, sometim es to improve their digestion, often to eliminate disulfide linkages from c omplicating analysis of the digest. After reduction, the sulfhydryls are us ually reacted with iodoacetamide (IAM), iodoacetic acid (IAA), or another e lectrophile to prevent reformation of disulfide linkages in a random manner . When the amount of protein may be reliably estimated, side reactions from excess IAM or IAA can be avoided. When this is not so, removal of excess i odoalkane can be accomplished by RPLC, by dialysis, or simply by allowing a reducing thiol to consume any excess. In mass spectrometric analysis of pr oteins isolated by 1D or 2D gels, removal of the excess iodoalkane is often accomplished simply by washing the gel prior to proteolytic digestion. Dur ing a recent study of the glutathionylation site mapping of actin, IAM was used to block any residual sulfhydryl groups remaining on the protein so th at they would not displace glutathione from its initial site. In addition, to avoid losses due to actin polymerization du-ring dialysis, the MI was al lowed to remain during the digestion. This further ensured that any sulfhyd ryl groups liberated during the digestion would be similarly blocked by the MI. Under these conditions, we observed the peptides to undergo N- as well as S-carbamidomethylation. In examining a series of other peptides alkylat ed with IAM in this way, we have found N-alkylation to be the rule rather t han the exception and even O-alkylation was detected. The main sites to whi ch the carbamidomethyl group attaches to the peptides have been located wit h LC-MS2 using an ion trap mass spectrometer and found to be the N-terminal amino group. A simple expedient to prevent such reactions when an excess o f reducing agent must be avoided is to run the alkylation in the presence o f a thioether such as 2,2 ' -thiodiethanol rather than a thiol.