Analysis of ligand binding by bioaffinity mass spectrometry

被引:4
作者
Zhu, Yusheng
Valdes, Roland
Simmons, Christine Q.
Linder, Mark W.
Pugia, Michael J.
Jortani, Saced A. [1 ]
机构
[1] Univ Louisville, Dept Pathol & Lab Med, Sch Med, Louisville, KY 40202 USA
[2] Univ Louisville, Dept Biochem & Mol Biol, Sch Med, Louisville, KY 40202 USA
[3] Bayer Healthcare LLC, Diagnost Business Grp, Elkhart, IN USA
关键词
Ligands analysis; SELDI-TOF; mass spectrometry; anti-digoxin antibody; urinary trypsin inhibitor; CYP450; 2E1; gene;
D O I
10.1016/j.cca.2006.02.023
中图分类号
R446 [实验室诊断]; R-33 [实验医学、医学实验];
学科分类号
1001 ;
摘要
Background: Ligand binding is commonly analyzed using various immumoassays that are generally time-consuming and some may require secondary antibodies or gel electrophoresis which are also time-consuming and sometimes subjective. We introduced various examples for a more rapid approach using pre-activated surface chips which are analyzed by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Specific applications presented in this study include immobilization of antigen, antibody or oligo DNA on preactivated chips with subsequent identification of the binding antibodies, antigens or DNA binding proteins to demonstrate the universal utility of this novel approach. Methods: BSA-digoxin conjugate (BSA-Dig), anti-digoxin antibody, anti-urinary trypsin inhibitor (uTi) antibody, or a double stranded oligo nucleotide based on the nucleotide sequence between -91 and -10 of the human CYP 450 2E1 promoter were immobilized on the Ciphergen preactivated surface chips. Anti-digoxin antibody, BSA-digoxin conjugate, uTi, and CYP450 2E1 promoter binding protein were captured on the chip and identified by SELDI-TOF MS. Results: A protein with 141 kDa was identified from anti-digoxin serum using BSA-Dig chips. This binding was competitively inhibited by addition of digoxin. Using anti-digoxin antibody, a peak at similar to 66kDa was detected in the preparation of BSA-Dig. This peak was also inhibited by free digoxin, suggesting BSA-Dig is detected. uTi fragments with similar to 3kDa to similar to 30kDa in the standard and urine samples were captured on the chip by anti-uTi antibody. Finally, we identified a 95-kDa CYP 450 2E1 promoter binding protein in HeLa cells nuclear extracts. Conclusions: Bioaffinity SELDI-TOF MS is a powerful and versatile approach for analysis of ligands. It eliminates tracer-labeled secondary antibodies and allows for determination of molecular weights of binding proteins and their ligands directly. This approach may also be considered for the detection of enzymes, receptors, or any other specific ligands. (c) 2006 Elsevier B.V. All rights reserved.
引用
收藏
页码:71 / 78
页数:8
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