Affinity Chromatography on Vancomycin Coupled to Reversed Phase Liquid Chromatography/ Electrospray-lon Trap Mass Spectrometry for the Screening of Combinatorial Libraries 2001, 54, 433-437 F. Lynen / E Borremans / P.Sandra* Department of Organic Chemistry, University of Gent, Krijgslaan 281 $4, 9000 Gent, Belgium; E-Maih pat.sandra@rug.ac.be Key Words Column liquid chromatography Affinity chromatography Vancomycin Mass spectroscopy detection Combinatorial chemistry Library screening Summary The combination affinity chromatography-mass spectrometry can be drastically improved by introducing a reversed phase column prior to the mass spectrometric detection. The interac- tions of the macrocyclic antibiotic vancomycin with ohgopeptides were used to illustrate the performance of the technique. A library of 36 peptides was successfully screened and the ac- tive compounds identified by electrospray MS(n). The strong affinity of compounds ending with (D)-alanine and with (D)-alanine or an aromatic (D)-amino acid in the penultimate position with vancomycin was confirmed. Introduction Since the introduction of affinity chroma- tography (AC) for the analysis and purifi- cation of bio-active compounds three dec- ades ago, the technique has gained in- creasing importance in the pharmaceuti- cal industry [1, 2, 3, 4]. Especially since the development of combinatorial chemistry, AC actively participates in the drug lead discovery process and is increasingly used as a tool for the screening of natural and synthetic mixtures. In early applications peptide combinatorial libraries could be screened successfully with AC whereby the retained and hence active compounds were collected and identified by peptide sequencing. Songyang et al. used the tech- nique by selecting specific phosphopep- tides out of sublibraries of a library of up to 5832 compounds for the interaction with an Src homology 2 (SH2) domain [5, 6]. Although accurate, sequencing has the drawbacks of long analysis times and the high quantities of pure compounds re- quired to obtain reliable results [7]. In more recent years, and as a logical exten- sion, AC has been used in combination with mass spectrometry (MS), equipped with atmospheric pressure ionization (API) sources such as electrospray (ESI) and, to a lesser extent, chemical ionization (APCI). This allowed the immediate iden- tification of retained compounds. Off-line AC-MS for the screening of a library was used by Huang et al. [8]. The direct combi- nation of affinity chromatography with ESI-MS could, however, only be achieved for a limited number of model separations [9]. The composition of the mobile phase normally used in AC does not give good ionization in MS. AC is conducted under aqueous conditions at a controlled pH and the use of buffers is required. The elu- tion of strongly interacting compounds needs a low pH, high ionic strengths and the addition of organic modifiers or chao- tropic agents to the mobile phase. These compositions lead to precipitation of salts in the ESI source, decreasing the sensitiv- ity of detection and the reproducibility of analysis. A solution can be found by using a second column in which the separation is performed under favorable ESI-MS conditions. This also increases the analyti- cal 'space' with the advantage that if sev- eral compounds are eluted together from the affinity column, they can be separated on the second column. Kelly et al. coupled AC with ESI-MS to screen a library of 361 peptides [10]. A small trap was used to remove interfering salts from the peptides prior to elution of the latter with a pH gra- dient to the MS. The receptor was the SH2 domain of phosphatidylinositol 3-ki- nase (PI 3-kinase), coupled to glutathione agarose as stationary phase. Kassel et al. used micro-column AC coupled to capil- lary reversed phase LC-ESI-MS for a vari- ety of applications [11]. Nedved et al. de- scribed an on-line immuno-affinity ex- traction-LC-LC-MS-MS method for the Original Chromatographia 2001, 54, October (No. 7/8) 433 0009-5893/00/02 433- 05 $ 03.00/0 9 2001 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH