Solution-Based Indirect Affinity Selection Mass Spectrometry—A

Article pubs.acs.org/ac

Solution-Based Indirect Affinity Selection Mass SpectrometryA General Tool For High-Throughput Screening Of Pharmaceutical Compound Libraries Thomas N. O’Connell,† Jason Ramsay,† Steven F. Rieth,‡ Michael J. Shapiro,† and Justin G. Stroh†,* †

Center of Chemistry Innovation and Excellence and ‡Research Informatics, Pfizer, Inc., Groton, Connecticut 06340, United States ABSTRACT: We show here that an automated solution-based affinity selection mass spectrometry (ASMS) system can be built exclusively from commercially available parts. The value of this technology lies in the throughput (∼1 × 105 compounds/day) coupled with a low hit rate. The system, being a binding assay, requires little development time yielding a fast timeline between target availability and hit identification. In addition, the use of exact mass simplifies the hit identification. We demonstrate this system using carbonic anhydrase as the target and a library of 144,000 proprietary compounds.

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tested in secondary assays requiring substantial time and finances. In contrast to this, solution-based indirect affinity selection mass spectrometry (ASMS) methods, which have been in operation since the mid 1990s6 holds the potential for rapidly screening large libraries, with much lower hit rates. Whereas most HTS assays are functional in nature resulting in biochemical turnover of substrate, ASMS assays are simple binding experiments and this is one cause for a lower hit rate. A compound must bind to the target and be detected by a mass spectrometer, but the fact that there is no functional readout means that there is also no interference from the functional readout system which typically involves a cascade reaction ultimately resulting in florescence. The major drawback of solution based indirect ASMS methods is that they are not commercially available. There are three different solution based indirect ASMS approaches, and these have been reviewed.7−9 All three work on the same basic principle of mixing a compressed set of small molecule compounds with a target, incubating the mixture, isolating the target with any small molecules bound to it, and ultimately analyzing the target/small molecule complex via LC/MS for the presence of the small molecules. While both LC/MS and large molecule isolation technologies are varied and widely available, the software needed to deconvolute the resulting mass spectral data has always been, at some level, proprietary, which has tended to limit the widespread use of these techniques. We have compared, by experiment, the three basic methods of solution based indirect ASMS and propose a complete ASMS

igh throughput screening (HTS) of various compound libraries has been a mainstay of Big-Pharma drug discovery since its introduction in the mid 1980s. Hallmarks of this technology are high-end automation and an ever increasing demand for quantity of compounds assayed per unit time, now upward of 1 × 106 compounds/day.1 However, the technology is not without its detractors. Criticism has focused on the quality of information obtained where the potential exists for a large number of false positive and false negative data points along with a very significant cost of operation. Without clear data suggesting which compounds would be valuable to pursue as drug candidates, many medicinal chemists have come to the conclusion that HTS does not provide sufficient quality information to warrant its expense and use at the beginning of the drug discovery process. This criticism is strong enough that managers of HTS facilities have felt it necessary to publish a defense of HTS as the method of choice for many if not most drug discovery programs.2 The two main quality issues surrounding HTS are false negative and false positive hits. On the one hand, false negative hits are at minimum disturbing since the absence of the hit can lead to a lost opportunity to pursue high quality chemical equity. Unfortunately, by its nature it is impossible to prove anything with negative data, and otherwise very hard to work with. On the other hand, false positive hits can be reduced using a variety of filters and tests.3 These filters have become quite critical to the HTS format as hit rates of 2−3% of compounds assayed yielding positive results are common, which then translates to tens of thousands of compounds to follow up on. The two basic mechanisms for identifying and removing false positives are plate based normalization (%inhibition) and complex statistical analysis.4,5 However, even with normalization and statistics, a large number of hits remain after filtering, which must then be © 2014 American Chemical Society

Received: March 13, 2014 Accepted: July 17, 2014 Published: July 17, 2014 7413

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Dulbecco’s phosphate buffered saline (PBS) buffer (Lonza, Walkersville, MD)was prepared. Twelve wells were prepared as follows: 40 μL of a 10 μM solution of Bovine Serum albumin (BSA) in PBS buffer was added to 1.6 μL DMSO solution of 400 pooled proprietary library compounds at 75 μM concentration each in a Greiner low volume 384-well plate. Final concentration: 10 μM BSA, 3 μM each compound, 4% DMSO. The plate was incubated for 40 min at ambient temperature with gentle shaking and then transferred to centrifuge at 4 °C. Plates were spun for 30 min at 2000 g to remove precipitates. Plates were then transferred to an HPLC auto sampler at 6 °C for ASMS analysis Positive and negative controls for Carbonic Anhydrase. A solution of 10 μM CA in PBS buffer was prepared and each of 7 known CA binders was prepared individually at 600 μM in DMSO. 0.1 μL of one standard was added to 20 μL of the CA solution for a final concentration of 10 μM CA, 3 μM standard, 0.5% DMSO. This was done separately for each binder creating 7 total positive controls and a separate 7 standards were diluted 0.1 μL in 20 μL of PBS to create negative controls. Ultrafiltration Method. A solution of 10 μM carbonic anhydrase (>80%, part no. C2522, Sigma-Aldrich, St. Louis, MO) in Dulbecco’s phosphate buffered saline (PBS) buffer (Lonza, Walkersville, MD) was prepared. The solution was divided into 200 μL aliquots and 1 μL of a DMSO (SigmaAldrich, St. Louis, MO) solution containing 600 μM of a known carbonic anhydrase inhibitor was added. Seven inhibitors were analyzed in separate wells. Final concentration: 10 μM CA, 3 μM compound, 4% DMSO. These aliquots were transferred to a 96well MultiScreen plate 10k NMWL Millipore Multiscreen filter plate (part no.MAUF01010, Millipore, Cork, Ireland.) Unused wells were loaded with 200 μL blue dye in PBS to observe variation in filtration throughout plate. The plate was allowed to incubate for 40 min at ambient temperature with gentle shaking with a 96-well receiver plate (part no. 650261, Greiner, Monroe, NC) underneath. The plates were then centrifuged together at 2000g for 65 min at 4 °C and the filtrate collected in the receiver plate was discarded. Twice more, 180 μL of PBS buffer was added to the wells and the plates were centrifuged at 2000g for 65 min at 4 °C with the filtrate collected being discarded each time. A solution of 1:1 water: acetonitrile with 0.1% formic acid (Fisher, Fair Lawn, NJ) was prepared and 180 μL of the solution was added to each well and incubated for 30 min to denature the protein. The plate together with a new 96-well receiver plate was centrifuged to dryness at 2000g for 99 min at 4 °C and the resulting filtrate wells were analyzed by UHPLC/MS on an Agilent Technologies (Wilmington, DE) 1200 UHPLC with a model 6220 TOF-MS using the same gradient conditions as described above. SpeedScreen Method. Novartis Speed Screen methodology was tested using a fresh 96-well MACROSpin P-2 SEC plate (part no. SNS P020L, The Nest Group, Southborough, MA). 400 μL of deionized water was added to each well and allowed to swell overnight at 4 °C. The plate, along with a receiver plate included in the kit, was then centrifuged for 2 min at 2000g at 4 °C to equilibrate, with the eluent in the receiver plate being discarded. This process was repeated twice more with 200 μL of water added before each centrifugation step to wash the gel. A loading plate consisting of an empty MACROSpin plate was placed atop the swelled SEC plate with a receiver plate underneath to create the full 3 piece assembly described in the literature.10,11 Twelve wells were prepared as follows: 40 μL of a 10 μM solution of carbonic anhydrase in PBS buffer was added to 1.6 μL DMSO solution of 400 pooled proprietary library

system that is significantly like one of the other systems, but is built exclusively from commercially available hardware and software components that can easily be assembled in any properly equipped mass spectrometry lab. The details are described below.

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EXPERIMENTAL SECTION Current Protocol. Experimental details can be easily varied depending on target. Typical procedure is as follows. Library pools containing 400 compounds at 75 μM each in DMSO are plated into Grenier (Monroe, NC) 7200 low volume plates, total volume of 200 nL per well. Plates are spun for 1 min at 2000 g at room temp. A solution of soluble target protein is prepared in appropriate buffer at 10 μM. Buffer contains any cofactors or reagents needed to optimize target binding. A typical buffer is 50 mM Tris or HEPES, pH = 7.4, 150 mM NaCl and 2 mM TCEP. Five microliters of protein solution is added to each well using a Formulatrix (Waltham, MA) Mantis plate dispensing robot. Final concentration: 10 μM protein, 3 μM each compound, 4% DMSO. Plates are incubated at room temp for 40 min with gentle shaking on a VWR (Radnor, PA) micro plate shaker and then transferred to Eppendorf (Hauppauge, NY) 5804 R centrifuge at 4 °C. Plates are spun for 30 min at 2000 g to remove precipitates. Plates are then transferred to an HPLC auto sampler at 6 °C for ASMS analysis. The ASMS system consists of two Agilent (Wilmington, DE) UHPLCs and one Agilent G6220 TOF mass spectrometer. The first UHPLC system is equipped with a 2.1 × 50 mm 5 μM 60Å PolyHydroxyEthyl A SEC column (PolyLC, Columbia, MD) using phosphate buffered saline solution (PBS) (Lonza, Walkersville, MD) as an isocratic mobile phase at 1.8 mL/min. The column is maintained at 4 °C. There are two diode array detectors used for the analysis, one before a divert valve and one after. At t = 0.10 min, the eluent containing protein and putative ligand binder is diverted via a 50 μL sample loop in a six way valve located in the flow path between the diode array detectors. The 50 μL slug is immediately introduced into the second UHPLC system equipped with a 2.1 × 50 mm C18 1.8 μ UHPLC column (at 60 °C and an Agilent G6220 TOF instrument operated in the positive ion mode. The mobile phase consists of (A) 0.1% formic acid (Fisher, Fair Lawn, NJ) with 10 mM ammonium formate and (B) 0.1% formic acid in 50:50 ACN/MeOH. Gradient A:B 98:2 hold for 0.6 min; ramp to 1:99 by 1.6 min, hold until 3.1 min; return to 98:2 by 3.3 min. The UHPLC eluent is diverted to waste from 0 to 0.6 min to avoid introducing salt into the MS source. The mass spectrometer is tuned and calibrated to manufacturer’s specifications prior to running each plate. Lock masses of 121.0509 and 922.0098 Da are infused during acquisition to maintain mass accuracy during the run. The SEC column is flushed with water containing 50% ACN from 0.10 to 1.10 min during the run and then equilibrated back with PBS for 2.2 min. This is done to remove any residual small molecules present. Binding ligands of interested are detected using APEX software (Sierra Analytics, Modesto, CA). Mass Spectral data from each well is compared to an SD file containing all known compounds in that well. A positive hit is characterized as one that has a protonated molecular ion of [M + H]+ and mono isotopic peak within 3 ppm of a known compound in the well. A peak smoothing of 4 scans is used. BSA Preparation. A solution of 10 μM Bovine Serum Albumin (>98%, part# A1470, Sigma-Aldrich, St. Louis, MO) in 7414

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Figure 1. Image of ultracentrifugation plate. Top plate: molecular weight cutoff filter plate. Bottom plate: receiving plate. Note the uneven liquid distribution well to well.

Figure 2. Flow diagram of the LC/SEC and UHPLC/HRMS detailing the components and shared flow path of the hybrid chromatography system used in ASMS screening.

compounds at 75 μM concentration each in a Greiner low volume plate. Seven of the wells had one of the known CA inhibitors spiked in at the same concentration. Final concentration: 10 μM CA, 3 μM each compound, 4% DMSO. The plate

was incubated for 40 min at ambient temperature with gentle shaking. 20 μL of the incubated solution was dispensed in row A of the prepared 3-piece plate and the other 20 μL was dispensed into the corresponding column in row B. The plate was chilled to 7415

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Figure 3. Example of data analysis software with extracted mass ion chromatogram, associated mass spectra, and plate/well scoring details for hit identification based on known contents of well.

4 °C and then centrifuged at 2000g for 1 min at 4C. The collected eluent in the receiver plate was then analyzed by UHPLC/MS on an Agilent Technologies (Wilmington, DE) 1200 UHPLC with a model 6220 TOF-MS using the same gradient profile as the other two techniques.

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therefore, we abandoned this technique. Additionally, this technique requires significant volume (200−400 μL) and hence very significant protein consumption. Also, it has been noted by Abbott that compound libraries need to be pooled by physiochemical properties. We then attempted the Novartis method,11,15,16 named “SpeedScreen”, which is based on separation using size exclusion gels. In this technique, compounds and target are incubated and then added to a gel manufactured in a 96-well plate format. The gel plate is spun, allowing the large molecular weight target with anything bound to it to elute the column first into a receiving plate. Once isolated, the receiving plate is analyzed via LC/MS for the presence of any small molecules. In our hands, this method works quite well and is amenable to automation. However, we have observed that a significant number of the wells in the plate show evidence of gel degradation causing breakthrough of small molecules which yields irreproducible data. This was observed even though plates were never reused. It has been noted in the literature11 that confirmation runs for this technique are to be run in triplicate. Based on the amount of breakthrough in the gels, we eventually abandoned this method as well. Additionally, the volume required for this assay was 25 μL or greater resulting in high protein consumption. Finally, we tested the Merck (NeoGenesis) system,17−21 named “ALIS” which stands for automated ligand identification system. Like SpeedScreen, ALIS relies on size exclusion gels, but the gels in ALIS are silica based HPLC columns allowing use of standard HPLC equipment. Using this system, we were able to

RESULTS

Solution-based ASMS techniques hold the promise of being high throughput devices that can produce data with a low hit rate. The three published procedures for generating solution based ASMS use LC/MS as the final detection system for determining which compounds in a mixture of compounds bound to the target. The largest differences are in the large molecule separation step. The first system tested was developed by van Breemen and coworkers12 and is in use at Abbott.13,14 In this method, a set of compounds is incubated with a large molecular weight target and the sample is then exposed to a molecular weight cutoff filter in an Eppendorf tube under centrifugation in order to separate the unbound small molecules from the target with anything bound to it. While we observed that this method actually worked, we were unable to determine how to make this operate in a truly plate based automated fashion. We attempted to run experiments in a plate based system using centrifugation as shown in Figure 1. As can be seen from the figure, there was no consistency between individual wells, hence the system was incapable of removing the solution with unbound small molecules equally or reproducibly, which again limited our ability to automate. Automation is a critical feature of executing high throughput technologies and, 7416

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where at least one compound was observed. There were a total of 92 hits observed in 88 different wells meaning that a few wells contained more than one hit. The upper chromatogram in the middle pane is the total ion current of the LC/MS for well K20 and the lower chromatogram in that pane is the single ion trace of the hit found in that well. The mass spectrum of that hit is shown in the bottom pane. The masses at 121, 622, and 922 are lock masses generated from an internal reference stream. The other major ion at 528.1717 Da. corresponds to the exact mass of the hit (expected mass = 528.1718 Da.; error = 0.25 ppm). The hit rate for this plate of a library at 0.06% (92/144,000) is typical in our experience. Of the 92 hits, 89 (97%) were sulfonamides and 80 (87%) were aryl sulfonamides, which is significant as the relationship between carbonic anhydrase and sulfonamides is well-known.22 It is important to note that the observed low hit rate in conjunction with an overall duty cycle time of approximately 5 min/well (yielding data on more than 1 × 105 compounds/day) makes this method reasonably attractive as a first pass screen for many drug targets. One reason for the low hit rate in this method is driven by the distinguishing features of true accurate mass analysis. It is both the selectivity and specificity of accurate mass determinations that leads to virtually no confusion between real analyte signal and chemical background. It must be acknowledged that while many manufactures of TOF instruments can produce data at the 1−2 ppm level in a high throughput manner, this is not universally true and, therefore, care should be taken to predetermine that the TOF used actually performs at this level. A second reason for low hit rate is driven by kinetics and not thermodynamics, and this fact is not particularly well appreciated. This method relies on compound staying bound to the target during the SEC purification step. During this process any compound bound to the target continues to break away from the complex with a particular rate constant of koff, but during this process there is no corresponding reforming of the complex as any small molecule compound coming off the complex is immediately captured by the SEC media, that is, kon = 0. Therefore, only compounds complexed with the target containing slower koff are observed. This tends to (but does not always) happen at tighter binding affinities. The fundamental relationship between thermodynamics (binding affinities) and kinetics (on and off rates) is well-known on first-principles, yet that relationship has been slightly obscured over a long period of time. Even though the concepts of residence time, and half-lives of drug-target complexes have long been known,23 still, there is a propensity among many scientists to perceive kinetic parameters as equivalent to thermodynamic ones. This often appears in the attempt to make KD values equivalent to, or at least linearly related to koff rate constants. Since KD = koff/kon, the assumption is made that kon is relatively constant over a series of compounds. If kon is relatively constant, then KD will track linearly with koff. Although it is well know that kon is often not relatively constant even within a specific series of compounds,24 the viewpoint still persists that it is. It is this misconception that leads to misunderstanding of ASMS data which is driven almost exclusively by koff rate constants. Sometimes we clearly observe compounds that have KD values in excess of 10 μM while also failing to observe other compounds with KD values in the midnanomolar range. Yet when the koff is taken into consideration, these results make sense. What we have developed here is an SEC method where target and any small molecule binder that is complexed with it are resident on the size exclusion column for approximately 5 s. In

successfully and reproducibly generate usable data. The hardware setup is shown in Figure 2. A compressed set of small molecule compounds (usually 400 compounds/well) is incubated with the target and loaded into a standard HPLC auto injector. Injections are made onto the size exclusion column, which is run at 4 °C to minimize the off-rate of any ligand bound complex. The target and any target/ligand complex elute the column in approximately 5 s thereby increasing the ability to capture ligands with relatively fast off rates. The target and target/ligand complex are heart-cut into a 50 μL loading valve which then becomes the injection valve of the UHPLC/MS system as shown. Upon exposure to the acidic environment of the UHPLC system the target is denatured and any small molecules that were bound to the target are liberated and therefore free to be observed using a standard small molecule LC/MS format. The critical difference with this system is that the method that we developed is based on accurate mass analysis. It is the selectivity of searching for accurate masses over extremely narrow mass ranges within the data set that allows us to perform this analysis using commercially and commonly available software. As a direct consequence of accurate mass analysis, there is no need to perform more than one analysis of each sample−there are no “before and after” comparisons of the data nor need for running analyses in duplicate or triplicate nor any other complex computational mechanism required in order to distinguish a real hit from a false positive. With accurate mass analysis a compound can be uniquely identified without need for orthogonal confirmation. Before an experiment can be performed, a proper library must be procured and formatted. We have a singleton library which has been developed with a widely encompassing view of compounds suitable for drug discovery. This library is highly curated. Sublibraries have been developed that represent the entire library with the number of distinct chemical entities ranging approximately 150 000−500 000 compounds. Sublibraries have been compressed to a level of 400 compounds/well. Since true accurate mass is involved in the final analysis of this method, the compression algorithm used paid no regard to the molecular weight of individual compounds other than to be aware of isomers present in any given well when the compounds were compressed into that well. Therefore, with regard to molecular weight, pooling was done randomly thereby reducing cost and effort. It is important to note that the quality of the compounds in the compressed libraries is critical to the success of this method because when using accurate mass as the guide, the software is only looking for the expected exact masses. If a compound was incorrectly labeled prior to the compression process or decomposed, it will not be observed in this method as it will have changed its exact mass. On the other hand, since one has the accurate mass (and by deduction the elemental composition) of the actual hits, there is a fairly high degree of confidence that the observed signal was real and was derived from the expected compound. It is well-known from the literature that the ALIS technique produces an extremely low false positive hit rate.7,17,18 Although our method is not identical in every respect to ALIS, there is substantial overlap between the two and we would expect that the technique we describe to be similar in this regard as well. As an example of this we ran a single plate (360 of 384 wells used) of a library containing 144,000 distinct compounds against carbonic anhydrase since this protein is well-known and lends itself to comparisons. The output from that plate is shown in Figure 3. The plate map shown in the upper left-hand quarter of Figure 3 shows red dots where a well contained no hits and green dots 7417

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Table 1. Literature Data for Seven Known Binders of Carbonic Anhydrasea

a

Data for standards 1, 5, 6, and 7 obtained from reference 24. Data for standards 2−4 obtained from reference 23. All data reported were obtained at 24° or 25° C.

order to perform this separation in 5 s, we approach SEC technology from a different viewpoint than that which is usually taken. The goal here is not to retain proteins on the column for separation from each other, but rather to have the protein elute in the void volume as quickly as possible and retain the unbound small molecules. Hence, we use columns with a pore size of 60 Å and column lengths 50 mm. Assuming that small molecule binders can still be observed after approximately 5 half-lives, we calculate that the minimum half-life of a target-binder complex to be approximately 1 s, which corresponds to a koff rate constant of approximately 0.7 s−1 or slower. Finally, we note that since ASMS provides data on compounds with relatively slow off-rates, it is expected that there should be relatively few hits observed in this assay assuming that a generally random library has been selected for the analysis. This is because there is a low probability that many compounds will have kinetics sufficient to remain bound through the SEC phase of the analysis. Therefore, a “high” hit rate (as opposed to other HTS assays) is a first warning sign that something has failed with the assay.

The method described here can produce both false positive and false negative information which can lead to confusion of results. Since this assay, by its nature, generates very few positive results, the proposed positive hits can be confirmed as true positive hits in other well-defined assays, either binding or functional in nature. One obvious exception to this method is in the analysis of targets that exhibit nonspecific binding. Examples of this would be GST fusion tags and serum albumin. We analyzed bovine serum albumin (BSA) with various library wells and found that the average number of “hits” per well containing 400 compounds was 7 (1.75%). This is at odds with an expected low hit rate (0.06% for carbonic anhydrase) which raises an immediate red flag in the analysis, alerting the user that the particular target is not appropriate for analysis via this method. False negative data, on the other hand, is much more difficult to deal with. There are a variety of sources for false negative data including but not limited to compounds bound to the wall of the well, compounds insoluble under the conditions used, compounds at lower concentration than expected, compounds 7418

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Figure 4. Normalized [M + H]+ single ion traces of all seven carbonic anhydrase standards listed in Table 1 using LC/MS only at 3 μM each component. Standards 4, 5, and 6 were not observed.

Figure 5. Normalized [M + H]+ single ion traces of all seven carbonic anhydrase standards listed in Table 1 using affinity selection mass spectrometry at 3 μM each component.

completely degraded, wrong compounds in the well to begin with, and ionization issues. Of the six potential issues listed, the first five are driven by the physiochemical quality of the library

used, and can be somewhat controlled by proper curation of the library, whereas the sixth (ionization issues) relates to a fundamental limitation of mass spectrometry. 7419

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(8) Shin, Y. G.; Van Breemen, R. B. Biopharm. Drug Dispos. 2001, 22, 353−372. (9) Siegel, M. M. Curr. Top. Med. Chem. (Hilversum, Neth.) 2002, 2, 13−33. (10) Brown, N.; Zehender, H.; Azzaoui, K.; Schuffenhauer, A.; Mayr, L. M.; Jacoby, E. J. Biomol. Screening 2006, 11, 123−130. (11) Zehender, H.; Mayr, L. M. Expert Opin. Drug Discovery 2007, 2, 285−294. (12) Johnson, B. M.; Nikolic, D.; van Breemen, R. B. Mass Spectrom. Rev. 2002, 21, 76−86. (13) Comess, K. M.; Schurdak, M. E.; Voorbach, M. J.; Coen, M.; Trumbull, J. D.; Yang, H.; Gao, L.; Tang, H.; Cheng, X.; Lerner, C. G.; McCall, J. O.; Burns, D. J.; Beutel, B. A. J. Biomol. Screening 2006, 11, 743−754. (14) Comess, K. M.; Trumbull, J. D.; Park, C.; Chen, Z.; Judge, R. A.; Voorbach, M. J.; Coen, M.; Gao, L.; Tang, H.; Kovar, P.; Cheng, X.; Schurdak, M. E.; Zhang, H.; Sowin, T.; Burns, D. J. J. Biomol. Screening 2006, 11, 755−764. (15) Brown, N.; Zehender, H.; Azzaoui, K.; Schuffenhauer, A.; Mayr, L. M.; Jacoby, E. J. Biomol. Screening 2006, 11, 123−130. (16) Muckenschnabel, I.; Falchetto, R.; Mayr, L. M.; Filipuzzi, I. Anal. Biochem. 2004, 324, 241−249. (17) Annis, D. A.; Athanasopoulos, J.; Curran, P. J.; Felsch, J. S.; Kalghatgi, K.; Lee, W. H.; Nash, H. M.; Orminati, J.-P. A.; Rosner, K. E.; Shipps, G. W.; Thaddupathy, G. R. A.; Tyler, A. N.; Vilenchik, L.; Wagner, C. R.; Wintner, E. A. Int. J. Mass Spectrom. 2004, 238, 77−83. (18) Annis, D. A.; Nazef, N.; Chuang, C.-C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495−15503. (19) Annis, D. A.; Nickbarg, E.; Yang, X.; Ziebell, M. R.; Whitehurst, C. E. Curr. Opin. Chem. Biol. 2007, 11, 518−526. (20) Annis, D. A.; Shipps, G. W., Jr.; Deng, Y.; Popovici-Mueller, J.; Siddiqui, M. A.; Curran, P. J.; Gowen, M.; Windsor, W. T. Anal. Chem. (Washington, DC, U. S.) 2007, 79, 4538−4542. (21) Annis, A.; Chuang, C.-C.; Nazef, N. Methods Princ. Med. Chem. 2007, 36, 121−156. (22) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146−189. (23) Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Nat. Rev. Drug Discovery 2006, 5, 730−739. (24) Swinney, D. C. Annu. Rep. Med. Chem. 2011, 46, 301−317 301 plate.. (25) Maren, T. H. Mol. Pharmacol. 1992, 41, 419−426. (26) Navratilova, I.; Papalia, G. A.; Rich, R. L.; Bedinger, D.; Brophy, S.; Condon, B.; Deng, T.; Emerick, A. W.; Guan, H.-W.; Hayden, T.; Heutmekers, T.; Hoorelbeke, B.; McCroskey, M. C.; Murphy, M. M.; Nakagawa, T.; Parmeggiani, F.; Qin, X.; Rebe, S.; Tomasevic, N.; Tsang, T.; Waddell, M. B.; Zhang, F. F.; Leavitt, S.; Myszka, D. G. Anal. Biochem. 2007, 364, 67−77.

To probe issues related to ionization efficiency, we analyzed seven known binders to carbonic anhydrase, listed in Table 1. All seven compounds were run using LC/MS at relevant concentrations (3 μM) to determine which compounds would give useable results before we attempted analysis via affinity selection. Results for those seven compounds are shown in Figure 4 which contains the normalized [M + H]+ single ion traces. Of the seven compounds tested, only four compounds were observed at the relevant concentrations. Of the four that were observed, only two (standards 7 and 2) gave a reasonable signal strength; the other two (standards 1 and 3) were very weak. Affinity selection mass spectrometry of all seven standards is shown in Figure 5, where again, the normalized [M + H]+ single ion traces yield information on only standards 7 and 2. Two very important features are demonstrated by these results. First, the ability to observe compounds as hits in this assay relies heavily on the ability to observe a sufficiently strong mass spectral signal of the native compound at relevant concentrations. Standards 4 and 5 do not ionize well enough to be observed at relevant concentrations in the affinity assay due to their small size, and Standard 6 is acidic and, therefore, does not ionize well in the positive ion mode; this despite the fact that all three standards have nanomolar binding affinities.25,26 Second, the binding affinities of the two standards observed (2 and 7) in the affinity assay vary by almost 5 orders of magnitude (see Table 1), yet their half-lives vary by only a factor of 35, and the fastest halflife is two seconds at room temperature, which is what allows these two standards to be seen.

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CONCLUSION Solution-based affinity selection mass spectrometry is a technology that holds much promise, for many reasons, including fast development time, high throughput capable, with a low hit rate. The major difficulty of this technique is that there are currently no commercial manufacturers of this technology. However, we have demonstrated that assembling a truly functioning HTS system of this type is well within the grasp of a fully functioning mass spectrometry lab.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: justin.stroh@pfizer.com. Notes

The authors declare no competing financial interest.

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dx.doi.org/10.1021/ac500938y | Anal. Chem. 2014, 86, 7413−7420