High Throughput Screening of High-Affinity Ligands for Proteins with Anion-Binding Sites using Desorption Electrospray Ionization (DESI) Mass Spectrometry

• Published in: Journal of the American Society for Mass Spectrometry 2014-03, Vol.25 (3), p.454-463
• Main Authors: Lu, Xin, Ning, Baoming, He, Dacheng, Huang, Lingyun, Yue, Xiangjun, Zhang, Qiming, Huang, Haiwei, Liu, Yang, He, Lan, Ouyang, Jin
• Format: Article
• Language: English
• Subjects: Affinity; Analytical Chemistry; Anions; Anions - analysis; Anions - chemistry; Anions - metabolism; Arid regions; Binding Sites; Bioinformatics; Biotechnology; Chemistry; Chemistry and Materials Science; Coordination compounds; Desorption; Displacement; Electrospraying; Glycoproteins; High-Throughput Screening Assays - methods; Ionization; Ions; Ligands; Mass spectrometry; Organic Chemistry; Protein Binding; Proteins; Proteins - chemistry; Proteins - metabolism; Proteomics; Research Article; Screening; Spectrometry, Mass, Electrospray Ionization - methods; Ultrafiltration
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1007/s13361-013-0753-3

A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein–ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α 1 -acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method. Figure ᅟ