Affinity selection MS

While the methods described in this chapter have been optimized for affinity selection-MS using continuous SEC, they are readily adaptable to spin-column, gel permeation, or other well validated and highly accessible two-stage AS-MS designs. The use of AS-MS for studying protein-ligand interactions, especially for the discovery of ligands from pools of compounds, has been reported by a number of experts in the pharmaceutical industry and academia over the past decade. 

D.A. Annis J. Athanasopoulos, P.J. Curran, J.S. Felsch, K. Kalghatgi, W.FI. Lee, H.M. Nash, J.-P.A. Orminati, K.E. Rosner, G.W. Shipps, Jr., G.R.A. Thaddupathy, A.N. Tyler, L. Vilenchik, C.R. Wagner, E.A. Wintner, An affinity selection-MS method for the identif cation of small molecule ligands from self-encoded combinatorial libraries. Discovery of a novel antagonist of E. coli dihydrofolate reductase, Int. J. Mass Spectrom., 238 (2004) 77. 

The schematic shown in Fig. 3.1 describes an optimized, integrated SEC-RPC-MS-based affinity selection platform developed at NeoGenesis, dubbed the... 

Several affinity screening methodologies that include MS-based readout and work under protein-excess conditions have been developed in the past decade [1]. Some examples include affinity selection/mass spectrometry (ASMS Abbott Labs [10]), size exclusion chromatography with LC-ESI-MS (see Chapter 2 and 3 [11-19]), the use of coupled or non-coupled pulsed ultra-filtration/mass spectrometry (summarized in this chapter [11, 20-23]), restricted access phase chromatography (see Chapter 5 [24, 25]), capillary electrophoresis [26, 27], target shift mass spectrometry [28], and multitarget affinity/specificity screening (MASS, see Chapter 10 [29, 30]). 

We have developed a high throughput ultrafiltration affinity screening method coupled to MS (affinity selection/mass spectrometry ASMS), which works with any soluble target and small molecule library (including natural products). ASMS is amenable to parallelization, efficient and robust enough to allow study... 

Combinatorial chemistry initiatives have created a tremendous challenge for activities that deal with the screening of these mixtures for activity against a specified target. MS-based approaches that use affinity selection, encoding methodologies, pulsed ultrafiltration, and anti-aggregatory approaches have been described. 

S. Kaur, L. McGuire, D. Tang, G. Dollinger, V. Huebner, Affinity selection and MS-based strategies to identify lead compounds in combinatorial libraries, J. Prot. Chem., 16(1997) 505. 

Figure 9 Affinity CE-MS (ACE) of a synthetic, all-D, Fmoc-DDXX library of 100 tetrapeptides using vancomycin as the receptor (A-D). Selected ion electropherograms for the masses are indicated (E) reconstructed ion electropherogram for runs without (left) and with (right) vancomycin in the electrophoresis buffer. Specific ACE and MS conditions are indicated elsewhere (87). (Reproduced with permission of the copyright holder, the publisher and the Journal of the American Chemical Society.)...

In the context of proteomic applications, microfluidic devices must comprise all functional elements necessary to perform essential processing steps prior to MS detection, i.e. sample clean-up, preconcentration, affinity selection, digestion and separation. As a result, micropumps, valves, mixers, microreactors,... 

Library Affinity Selection-Mass Spectrometry (LAS-MS) In this approach the library components are reacted with agarose gel-bound receptor in solution [101]. After washing the beads, peptides of increasing receptor affinity are released by adding to the beads elution buffers of different pH values the peptides released are identified with ESI-MS or ESI-MS/MS. 

Lingeman, H., and Irth, H. (2009) Online magnetic bead dynamic protein-affinity selection coupled to LC-MS for the screening of pharmacologically active compounds. Analytical Chemistry, 81 (11), 4263-4270. 

Immunosorbents have also found applicability in on-line SPE analysis. An antibody is immobilized on to a silica support and used as an affinity ligand to retain targeted analytes. Components not recognized by the antibody are not retained and some degree of selectivity is attained. Recoveries of 87-103% were obtained for atrazine, simazine, DEA, propazine, and terbuthylazine at the 0.2 xgL concentration level when using immunosorbent SPE (80 mg silica and 2 mg anti-atrazine and anti-chlortoluron antibodies) on-line with LC/APcI-MS however, this method is not applicable to DIA (0% recovery). This compound may be better retained when using an... 

The first step in biopharmaceutical development is the selection of a clone in a specific cell line. Whole-mass analysis, if possible, is a fairly simple and powerful tool at this stage to verify the successful expression and translation of the desired protein. VanAdrichem et al.65 described the use of MALDI MS to monitor protein expression in several mammalian cell lines like CHO DXB11, CHO SSF3, and hybridomas. Quantitative MALDI-TOF MS measurements of an IgG antibody and insulin during large-scale production in hybridoma cells were comparable to affinity chromatography results. 

---