Info

Advantages of GPC Spin Columns

A spin column is a short column packed with GPC media that is centrifuged (see Fig. 2.1A, B). The media used for GPC are also referred to as size exclusion chro-matography and gel filtration chromatography media. The gel and sample are prepared with buffers compatible with processing the protein-drug complex in its native state. Upon loading the sample at the top of the column and centrifuga-tion of the column, the lower molecular weight (MW) free ligands are separated rapidly from the higher MW protein and protein-ligand complexes. The free li-gands are unfractionated and retained by the gel while the eluate, corresponding to the solvent front, passes unrestricted through the gel containing the protein and protein-ligand complexes. The GPC spin column eluate is then denatured

Fig. 2.1 GPC spin column used for isolating protein/RNA-drug non-covalent complexes in the eluate upon centrifugation and the ESI-MS steps to detect the ligands upon denaturing of the protein/RNA-drug non-covalent complexes. (A) Spin column cartoon, (B) Photo of a miniature GPC spin column, (C) Schematic of GPC spin column/ESI-MS procedures.

Fig. 2.1 GPC spin column used for isolating protein/RNA-drug non-covalent complexes in the eluate upon centrifugation and the ESI-MS steps to detect the ligands upon denaturing of the protein/RNA-drug non-covalent complexes. (A) Spin column cartoon, (B) Photo of a miniature GPC spin column, (C) Schematic of GPC spin column/ESI-MS procedures.

and the free ligand is analyzed by flow-injection analysis or HPLC using ESI-MS under denaturing conditions (see Fig. 2.1C.) This procedure decouples the preparation (incubation), separation and analysis steps so that each step can be individually optimized in a flexible fashion. The methodology is simple to apply and rapid to implement and utilizes standard size exclusion and ESI mass spectrom-etry techniques under optimum conditions for sample preparation, isolation, detection, quantitation and automation.

An example of the GPC spin column/ESI-MS methodology for drug screening is illustrated in Fig. 2.2 for identifying a non-covalently bound inhibitor to a protein target. Figure 2.2A displays the ESI mass spectrum of an impure peptidic di-fluoromethyl ketone inhibitor (DFMK) before passing through the GPC spin column. Figure 2.2B displays the ESI mass spectrum after passage through the GPC spin column of the incubated mixture of impure DFMK with cytomegalovirus protease (CMVP). The impure inhibitor, upon passing through the GPC spin column, emerged as a purified major component together with the protease with which it formed a non-covalent complex. The gel retained all other impurity components. In this way, large numbers of drug candidates can be routinely screened with a protein target because the non-covalently bound drug candidates pass

Fig. 2.2 ESI mass spectra obtained from the GPC spin column/ESI-MS screening assay of non-covalently bound protease-inhibitor complexes. Enzymatically active CMVP A144D/C87A/C138A/C161A was used in this experiment. (A) Reference ESI mass spectrum of impure inhibitor DFMK (MW 988.5 Da). (B) ESI mass spectrum of the spin column eluate of CMVP A144D/C87A/ C138A/C161A and DFMK, incubated at a

Fig. 2.2 ESI mass spectra obtained from the GPC spin column/ESI-MS screening assay of non-covalently bound protease-inhibitor complexes. Enzymatically active CMVP A144D/C87A/C138A/C161A was used in this experiment. (A) Reference ESI mass spectrum of impure inhibitor DFMK (MW 988.5 Da). (B) ESI mass spectrum of the spin column eluate of CMVP A144D/C87A/ C138A/C161A and DFMK, incubated at a molar ratio of 1: @10. (C) ESI mass spectrum of the microconcentrator filtrate (3 kDa cutoff centrifugal ultrafiltration membrane) obtained under denaturing conditions (3% acetic acid in 1:1 water:acetonitrile, v:v) from the non-covalently bound complex of CMVP A144D/ C87A/C138A/C161A and DFMK generated from the GPC spin column eluate. Reprinted from reference [13] with permission from John Wiley & Son.

through the GPC spin column and are detected by ESI-MS while all the other drug candidates are retained by the GPC spin column and are not detected.

Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook


Post a comment