蛋白质组学

关于为什么烷基化后PMF检索要选carbamidomethyl(C)

不知道大家是否清楚,我特地去查了一下原始文献,摘要中就有说明

mascot中关于Iodoacetamide(碘乙酰胺) derivative的链接
http://www.unimod.org/cgi/unimod.cgi?sort_field1=mono_mass&sort_field2=full_name&first_record_to_display=0&records_per_page=25&session_id=UT1DESR4B7YVKXMO&record_id=4&columns_to_view=full_name&columns_to_view=code_name&columns_to_view=mono_mass&columns_to_view=avge_mass&columns_to_view=composition&display_details_view.x=3&display_details_view.y=10

参考文献
Anal. Chem., 73 (15), 3576 -3582, 2001. 10.1021/ac0103423 S0003-2700(01)00342-0

Not subject to U.S. Copyright. Published 2001 American Chemical Society
Overalkylation of a Protein Digest with Iodoacetamide

Emily S. Boja and Henry M. Fales*

National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Drive, MSC 1676, Bethesda, Maryland 20892

Received for review March 22, 2001. Accepted May 22, 2001.

Abstract:

Cystine linkages in proteins are often opened with reducing agents, sometimes to improve their digestion, often to eliminate disulfide linkages from complicating analysis of the digest. After reduction, the sulfhydryls are usually reacted with iodoacetamide (IAM), iodoacetic acid (IAA), or another electrophile 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 iodoalkane can be accomplished by HPLC, by dialysis, or simply by allowing a reducing thiol to consume any excess. In mass spectrometric analysis of proteins isolated by 1D or 2D gels, removal of the excess iodoalkane is often accomplished simply by washing the gel prior to proteolytic digestion. During a recent study of the glutathionylation site mapping of actin, IAM was used to block any residual sulfhydryl groups remaining on the protein so that they would not displace glutathione from its initial site. In addition, to avoid losses due to actin polymerization during dialysis, the IAM was allowed to remain during the digestion. This further ensured that any sulfhydryl groups liberated during the digestion would be similarly blocked by the IAM. Under these conditions, we observed the peptides to undergo N- as well as S-carbamidomethylation. In examining a series of other peptides alkylated with IAM in this way, we have found N-alkylation to be the rule rather than the exception and even O-alkylation was detected. The main sites to which the carbamidomethyl group attaches to the peptides have been located with 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 of reducing agent must be avoided is to run the alkylation in the presence of a thioether such as 2,2'-thiodiethanol rather than a thiol.

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