N-isopropyliodoacetamide in the reduction and alkylation of proteins: use in microsequence analysis.
A new reagent, N-isopropyliodoacetamide (NIPIA), for alkylation of sulfhydryl groups on proteins for microdigestion and microsequencing is described. The utility of this reagent in both of these procedures has been demonstrated. NIPIA was shown to be especially useful in microsequence analysis, where it yields high sensitivity in detection of Cys residues. This is because the phenylthiohydantoin (PTH) derivative of NIPIA-alkylated cysteine [PTH-Cys(NIPCAM)] appears as a sharp peak in a standard reverse-phase HPLC analysis of PTH amino acids, and elutes between PTH-Tyr and PTH-Pro where no other peaks are present. Thus the use of NIPIA circumvents various problems associated with HPLC analysis of PTH-Cys when other commonly used agents are employed for sulfhydryl alkylation, such as coeluting peaks or low signal levels. Procedures for the synthesis of NIPIA and other analogs, as well as PTH-Cys(NIPCAM), are also presented, and HPLC retention times for their corresponding PTH-Cys derivatives are compared.
Krutzsch
H C
HC
Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Maryland.
Inman
J K
JK
D000478:Alkylation; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis; D007202:Indicators and Reagents; D007460:Iodoacetamide / Q000031:analogs & derivatives*; D008832:Microchemistry / Q000379:methods; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010669:Phenylthiohydantoin; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D017421:Sequence Analysis / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000378:metabolism
D007202:Indicators and Reagents; D011506:Proteins; D013438:Sulfhydryl Compounds; D010669:Phenylthiohydantoin; C080491:N-isopropyliodoacetamide; D003545:Cysteine; D007460:Iodoacetamide