Capillary zone electrophoresis of Cowpea mosaic virus and peak identification.
Cowpea mosaic virus (CPMV) was analyzed by CZE and the two separated subcomponents recorded at approximately 2.2 and approximately 2.7 min were respectively designated to be the slow electrophoretic form of CPMV (CPMV(s)) and the fast electrophoretic form of CPMV (CPMV(f)), the two electrophoretic forms of CPMV, by the following methods: first, CZE analysis of the biospecific complexes of CPMV and anti-CPMV polyclonal antibody proved the properties of CPMV; second, isolation and CZE analysis of CPMV(f) and CPMV(s). Because CPMV(s) was precipitated and CPMV(f) was left in the supernatant at pH 5.6, the only one peak at approximately 2.7 min from the supernatant was assigned for CPMV(f), while the peak at approximately 2.2 min dominating for the precipite was deemed to be CPMV(s). Third, viral characterization by directly analyzing the capsid proteins using MALDI-TOF-MS. Based on the theoretical molecular weights calculated from the sequence of the capsid proteins, the three peaks at m/z 21 516.75, 23 745.8 and 42 509.22 for natural CPMV were destined for the small (S) protein of CPMV(f), the S protein of CPMV(s) and the large (L) protein of both of them, respectively. As expected, the non-appearance of the peak at m/z 23 745.8 for the isolated CPMV(f) sample indicated the absence of CPMV(s), and the peak at m/z 23 745.8 was predominant in the spectrum for the precipitated CPMV(s) sample. After the confirmation of CPMV(s) and CPMV(f), the CZE separation of them was optimized. The developed analysis method has proven useful in investigating the stability of CPMV and the effect of 2,4-dinitrophenyl-decoration on the modification of the electrophoretical behavior of CPMV.
Liang
Shucai
S
Department of Pharmaceutical Analysis, College of Pharmacy, Wuhan University, Wuhan, PR China.
Schneider
Rudolf J
RJ