摘要
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1 h后,采用高效液相色谱-激光诱导荧光检测法(HPLC-LIF)分析(λex=473 nm,λem=525 nm),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。
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）（λex=473nm,λem = 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 LIF 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.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2011年第7期1027-1032,共6页
Chinese Journal of Analytical Chemistry
基金
国家重点基础研究发展规划(973)项目(No.2011CB910400)
上海市科委专项基金(No.10dz2220500)
华东理工大学优秀青年教师基金(No.YJ0157/45)资助项目
关键词
蛋白
多肽
激光诱导荧光
高效液相色谱
Proteins
Peptides
Laser-induced fluorescence
High performance liquid chroma-tography
