Analysis of ligand binding by bioaffinity mass spectrometry.
BACKGROUND
Ligand binding is commonly analyzed using various immunoassays 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 pre-activated 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 pre-activated 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 141kDa 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 approximately 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 approximately 3kDa to approximately 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.
Zhu
Yusheng
Y
Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY 40202, USA.
Valdes
Roland
R
Simmons
Christine Q
CQ
Linder
Mark W
MW
Pugia
Michael J
MJ
Jortani
Saeed A
SA
D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004077:Digoxin / Q000032:analysis / Q000276:immunology; D006801:Humans; D008024:Ligands; D015345:Oligonucleotide Probes / Q000032:analysis / Q000235:genetics; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014361:Trypsin Inhibitors / Q000032:analysis / Q000276:immunology
D000906:Antibodies; D004268:DNA-Binding Proteins; D008024:Ligands; D015345:Oligonucleotide Probes; D011506:Proteins; D014361:Trypsin Inhibitors; D004077:Digoxin; D003577:Cytochrome P-450 Enzyme System