Fluorescent labeling of cysteinyl residues. Application to extensive primary structure analysis of proteins on a microscale.
The specificity and efficiency of fluorescent labeling of proteins by reduction and subsequent alkylation with 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS) [J.J. Gorman, (1987) Anal. Biochem. 160, 376-387] has been investigated. Proteins studied include porcine insulin, chicken ovalbumin and bovine serum albumin. Amino acid analysis of the B-chain derivative of insulin revealed quantitative recovery of cysteine in its S-carboxymethyl form and no other carboxymethylated amino acid derivatives. Fast-atom-bombardment mass spectrometric (FAB-MS) analysis of this derivative also indicated specific labeling of cysteine residues and automated stepwise protein sequence analysis of the derivative was performed to completion with initial and average repetitive yields of 73% and 96%, respectively. Tryptic peptides produced from the ovalbumin and serum albumin derivatives were fractionated by HPLC and subsequently analysed by amino acid analysis, FAB-MS and automated stepwise protein sequence analysis. These analyses have revealed that the labeling procedure exhibits a high degree of efficiency and is specifically directed towards S-alkylation of cysteine residues. The high level of fluorescence intensity of the label enabled specific detection of trace quantities of cysteine-containing peptides derived from contaminating protein(s). It is apparent that in addition to facilitating isolation of small quantities of proteins the labeling procedure is compatible with standard protein chemistry techniques involved in obtaining extensive structural data on isolated proteins.
Gorman
J J
JJ
Commonwealth Scientific and Industrial Research Organisation, Australian Animal Health Laboratory, Geelong.
Corino
G L
GL
Mitchell
S J
SJ
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis*; D006868:Hydrolysis; D007328:Insulin / Q000032:analysis; D008832:Microchemistry; D009282:Naphthalenesulfonates; D010047:Ovalbumin / Q000032:analysis; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis; D013050:Spectrometry, Fluorescence
D000345:Affinity Labels; D000596:Amino Acids; D007328:Insulin; D009282:Naphthalenesulfonates; D010455:Peptides; D011506:Proteins; D012710:Serum Albumin, Bovine; C006422:1,8-I-AEDANS; D010047:Ovalbumin; D003545:Cysteine