Overalkylation of a Protein Digest with Iodoacetamide
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- 作者：Emily S. Boja and Henry M. Fales
- 刊名：Analytical Chemistry
- 出版年：2001
- 出版时间：August 1, 2001
- 年：2001
- 卷：73
- 期：15
- 页码：3576 - 3582
- 全文大小：148K
- 年卷期：v.73,no.15(August 1, 2001)
- ISSN：1520-6882
文摘
Cystine linkages in proteins are often opened with reducing agents, sometimes to improve their digestion, oftento eliminate disulfide linkages from complicating analysisof the digest. After reduction, the sulfhydryls are usuallyreacted with iodoacetamide (IAM), iodoacetic acid (IAA),or another electrophile to prevent reformation of disulfidelinkages in a random manner. When the amount ofprotein may be reliably estimated, side reactions fromexcess IAM or IAA can be avoided. When this is not so,removal of excess iodoalkane can be accomplished byHPLC, by dialysis, or simply by allowing a reducing thiolto consume any excess. In mass spectrometric analysisof proteins isolated by 1D or 2D gels, removal of theexcess iodoalkane is often accomplished simply by washing the gel prior to proteolytic digestion. During a recentstudy of the glutathionylation site mapping of actin, IAMwas used to block any residual sulfhydryl groups remaining on the protein so that they would not displaceglutathione from its initial site. In addition, to avoid lossesdue to actin polymerization during dialysis, the IAM wasallowed to remain during the digestion. This furtherensured that any sulfhydryl groups liberated during thedigestion would be similarly blocked by the IAM. Underthese conditions, we observed the peptides to undergoN- as well as S-carbamidomethylation. In examining aseries of other peptides alkylated with IAM in this way,we have found N-alkylation to be the rule rather than theexception and even O-alkylation was detected. The mainsites to which the carbamidomethyl group attaches to thepeptides have been located with LC-MS2 using an ion trapmass spectrometer and found to be the N-terminal aminogroup. A simple expedient to prevent such reactions whenan excess of reducing agent must be avoided is to run thealkylation in the presence of a thioether such as 2,2'-thiodiethanol rather than a thiol.