Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography-digestion-liquid chromatography-electrospray mass spectrometry.
A multidimensional analytical method for the rapid determination and identification of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography-electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the different pretreatment steps was performed. The automated system was tested for the identification of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identification by a total sequence coverage of approximately 40%.
Carol
Jeroen
J
Department of Analytical Chemistry and Applied Spectroscopy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. j.carol@few.vu.nl
Gorseling
Maarten C J K
MC
de Jong
Camiel F
CF
Lingeman
Henk
H
Kientz
Charles E
CE
van Baar
Ben L M
BL
Irth
Hubertus
H
D000595:Amino Acid Sequence; D002772:Cholera Toxin / Q000032:analysis*; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004768:Enterotoxins / Q000032:analysis*; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization
D004768:Enterotoxins; D011506:Proteins; C031818:enterotoxin B, staphylococcal; D002772:Cholera Toxin