Two-dimensional turbulent flow chromatography coupled on-line to liquid chromatography-mass spectrometry for solution-based ligand screening against multiple proteins.
We present herein a novel bioseparation/chemical analysis strategy for protein-ligand screening and affinity ranking in compound mixtures, designed to increase screening rates and improve sensitivity and ruggedness in performance. The strategy is carried out by combining on-line two-dimensional turbulent flow chromatography (2D-TFC) with liquid chromatography-mass spectrometry (LC-MS), and accomplished through the following steps: (1) a reversed-phase TFC stage to separate the protein/ligand complex from the unbound free molecules, (2) an on-line dissociation process to release the bound ligands from the complexes, and (3) a second mixed-mode cation-exchange/reversed-phase TFC stage to trap the bound ligands and to remove the proteins and salts, followed by LC-MS analysis for identification and determination of the binding affinities. The technique can implement an ultra-fast isolation of protein/ligand complex with the retention time of a complex peak in about 5s, and on-line prepare the "clean" sample to be directly compatible with the LC-MS analysis. The improvement in performance of this 2D-TFC/LC-MS approach over the conventional approach has been demonstrated by determining affinity-selected ligands of the target proteins acetylcholinesterase and butyrylcholinesterase from a small library with known binding affinities and a steroidal alkaloid library composed of structurally similar compounds. Our results show that 2D-TFC/LC-MS is a generic and efficient tool for high-throughput screening of ligands with low-to-high binding affinities, and structure-activity relationship evaluation.
Zhou
Jian-Liang
JL
Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Ministry of Education, Nanjing, China.
An
Jing-Jing
JJ
Li
Ping
P
Li
Hui-Jun
HJ
Jiang
Yan
Y
Cheng
Jie-Fei
JF
D000110:Acetylcholinesterase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000470:Alkaloids; D002091:Butyrylcholinesterase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000032:analysis / Q000378:metabolism; D002802:Cholinesterases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D004867:Equipment Design; D005702:Galantamine / Q000032:analysis / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D019151:Peptide Library; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012717:Sesquiterpenes / Q000032:analysis / Q000378:metabolism; D013329:Structure-Activity Relationship
D000470:Alkaloids; D002800:Cholinesterase Inhibitors; D008024:Ligands; D019151:Peptide Library; D011506:Proteins; D012717:Sesquiterpenes; C050426:huperzine A; D005702:Galantamine; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase; D002802:Cholinesterases