高效液相色谱法紫外与激光诱导荧光检测蛋白质的分析对比

摘要

The protein sample extracted from rat liver tumor tissues and its enzymic hydrolysates were rndetected by high performance liquid chromatography-laser induced fluorescence detector(HPLC-LIF) rn(λex = 473nm, λem = 525 nm) after labeled with 4-fluoro-7-nitro-2 , 1, 3-benzoxadiazole ( NBDF) in rndark at room temperature for 1 h with borate buffer (50 mmol/L, pH=8. 5) as reaction medium, and rnthe results were compared with those obtained from HPLCUV (at 214nm) without derivatization.rnThe analysis of enzymic hydrolysates shows that LIF detector can greatly improve the detection by its rnhigh sensitivity: the peak intensities of most components are 10 times higher than UV detection and rn42 peaks more were observed. For the analysis of protein sample, most of the peaks detected by UV rnare located in strong polarity area, which indicates that these components with strong polarity have rnhigh UV absorption. The signals obtained from LIF are located in weak polarity or non-polarity area.rnOne reason is that derivatization can change the polarity of the components, and the other is that comrnponents with weak or non-polarity might be labeled more easily. This result also suggests selectivity rndifference between these two methods over the real protein sample. In addition, selective label of rncomplex sample by changing reaction conditions (such as pH and reaction media) enables to achieve rnhigh sensitive detection of low abundance proteins and peptides with better selectivity.%将鼠肝肿瘤组织蛋白及其酶解产物经4-氟-7-硝基-2,1,3-苯并氧杂恶二唑(NBD-F)在50 mmol/L硼砂缓冲溶液(pH 8.5)中室温避光衍生1h后,采用高效液相色谱-激光诱导荧光检测法(HPLC-LIF)分析(λ(ex)=473nm,λ(em)=525 mn),并与高效液相色谱-紫外检测法(HPLC-UV)在检测波长214 nm所得结果进行对比.对蛋白质酶解产物的分析结果表明,LIF检测器因其更高的检测灵敏度,可显著提高检测能力,多数色谱峰较紫外信号高10倍以上,最终多检测到42个峰.进一步对鼠肝肿瘤组织蛋白提取液的检测结果进行对比,HPLC-UV法检测到的组分集中在强极性区域,说明样品中极性组分的紫外吸收较强;HPLC-LIF法检测到的组分更多集中在弱极性和非极性区域,一方面因为衍生化能够改变组分的极性,另一方面因为弱极性和非极性组分更容易被衍生化,此结果也说明两种方法对样品的检测选择性存在明显差异.通过改变荧光衍生化反应条件(溶液pH值、缓冲液介质)对复杂样品进行选择性标记,有利于实现对低丰度蛋白和多肽的选择性检测.