Comparative Study on Determination of Proteins by High Performance Liquid Chromatography with Ultraviolet or Laser-Induced Fluorescence as Detector

The protein sample extracted from rat liver tumor tissues and its enzymic hydrolysates were detected by high performance liquid chromatography-laser induced fluorescence detector(HPLC-LIF) (lambda(ex) = 473nm, lambda(em) = 525 nm) after labeled with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) in dark at room temperature for 1 h with borate buffer (50 mmol/L, pH = 8.5) as reaction medium, and the results were compared with those obtained from HPLC-UV (at 214nm) without derivatization. The analysis of enzymic hydrolysates shows that LIP detector can greatly improve the detection by its high sensitivity: the peak intensities of most components are 10 times higher than UV detection and 42 peaks more were observed. For the analysis of protein sample, most of the peaks detected by UV are located in strong polarity area, which indicates that these components with strong polarity have high UV absorption. The signals obtained from LIF are located in weak polarity or non-polarity area. One reason is that derivatization can change the polarity of the components, and the other is that components with weak or non-polarity might be labeled more easily. This result also suggests selectivity difference between these two methods over the real protein sample. In addition, selective label of complex sample by changing reaction conditions (such as pH and reaction media) enables to achieve high sensitive detection of low abundance proteins and peptides with better selectivity.