Optimization of sample recovery from the nitrocellulose support used in plasma desorption mass spectrometry and its use for multiple analyses of insulin.
The parameters for recovery of sample from the nitrocellulose support used in plasma desorption are optimized. The losses in washing, in situ reactions, and extraction procedures are quantitatively evaluated. At least 80% of the sample can be effectively extracted or transferred to a polyvinyl difluoride membrane with 2-propanol-water mixture in the range 1:1-2:3 v/v. Quantitative losses of insulin during washing procedures vary from 0 to 50% depending on the washing procedure used. The losses in in situ reactions are negligible. Optimization of the procedures allows several successive procedures to be carried out after adsorption of 1 nmol of insulin on the nitrocellulose support. These include in situ reaction, extraction of A and B chains followed by S-alkylation, chain separation by high-performance liquid chromatography, mass spectrometric analysis of the separated chains, and finally automatic sequence after transfer to the sequenator.
Jespersen
S
S
Department of Molecular Biology, Odense University, Denmark.
Talbo
G
G
Roepstorff
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003101:Collodion; D003545:Cysteine / Q000032:analysis; D004229:Dithiothreitol; D006801:Humans; D007328:Insulin / Q000032:analysis*; D007457:Iodine Radioisotopes; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011145:Polyvinyls
D007328:Insulin; D007457:Iodine Radioisotopes; D008567:Membranes, Artificial; D011145:Polyvinyls; C077776:polyvinyl fluoride; D003101:Collodion; D003545:Cysteine; D004229:Dithiothreitol