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46and further demonstrate the capability of the platform in avalidation study screening 23,000 small molecules on asingle day.Materials and MethodsMaterialsDisposable HTS MALDI target plates (no. 1847006) andHTS MALDI Adapter (no. 8283496) were purchased fromBruker Daltonics (Billerica, MA). 384-well polypropylene(PP) microwell plates (no. 784201) were obtained fromGreiner Bio-One (Frickenhausen, Germany). Recombinanthuman PTP1B CA (PTP1B C215A h2-321 NHis8TEV)was purchased from Trenzyme GmbH (Konstanz,Germany). The catalytic domain of PTP1B (PTP-1Bh2-321 NHis8TEV) was produced in-house at BoehringerIngelheim. α-Cyano-4-hydroxycinnamic acid (4-HCCA;no. 70990), acetonitrile (no. 34851), DMSO (no. D5879),ammonium acetate (no. 73594), bovine gamma globulin(BGG; no. G5009), HEPES (no. H3375), isopropanol (no.34863), and SYPRO Orange (no. S5692) were obtainedfrom Sigma Aldrich (St. Louis, MO). Trifluoroacetic acid(TFA; no. 6957) was purchased from Carl Roth (Karlsruhe,Germany). Bovine serum albumin (BSA; no. 11945.03) anddithiothreitol (DTT; no. 20710.04) were obtained fromServa (Heidelberg, Germany). Bio-Gel P10 Media (Fine:45–90 µm; no. 150-4144) was purchased from BioRad(Feldkirchen, Germany). Multiscreen HTS 384-well filterplates [0.45 µm polyvinylidene fluoride (PVDF) membrane; no. MZHVN0W10], Tris (no. 108382), and MgCl2*6H2O (no. 105833) were obtained from Merck Millipore(Burlington, MA). Peptides (custom synthesis), KCl 2Msolution (no. AM9640G), and TCEP [tris(2-carboxyethyl)phosphine; no. 20491] were purchased from Thermo FisherScientific (Waltham, MA). AlphaScreen reagents (Strepta vidin Donor beads, no. 676002; and anti-6xHis AlphaLISAAcceptor beads, no. AL128M) were obtained fromPerkinElmer (Waltham, MA).MALDI-TOF AS-MS Screening WorkflowThe setup of incubation mixtures, the in-plate SEC-basedseparation process, and the transfer of 384-well plates into1536-well format were performed on a fully automated system featuring a modular design and robotic plate transportation. At the beginning of each iterative cycle, a 384-wellcompound plate was transported to a CyBio Well vario liquid-handling system (Analytik Jena, Jena, Germany)equipped with a 384-well format capillary head to transfer50 nL of 5 µg/µL or 10 mM compound solutions or DMSOto the wells of a 384-well PP assay plate. The compoundplate was then put back into storage, and the assay plate wastransported to a Thermo Multidrop Combi (Waltham, MA)SLAS Discovery 26(1)dispenser, where 5 µL of 10 µM (0.40 mg/mL) PTP1B CAin 50 mM HEPES (pH 7.4), 100 mM NaCl, 2 mM DTT,0.01% BGG, 0.001% Tween20, and 25 µM quality control(QC) peptide (assay buffer) were added to every well. Theassay plate was then vortexed for 30 s at 1000 rpm on aBioshake 3000 (Quantifoil Instruments, Jena, Germany)device, covered with a plastic lid, and incubated at 24 C for40 min in a humidified chamber. Just before the completionof the incubation step, a loaded 384-well filter plate wasstacked onto an empty 384-well PP plate, and both wereloaded into a centrifuge. After centrifugation for 2 min at800 rcf, the PP plate was discarded, and the filter plate wasplaced in a CyBio Well vario liquid-handling system(Analytik Jena) equipped with a 384-well format and 40 µLhead. After transportation of the assay plate to the multichannel pipettor, incubation solutions were quantitativelytransferred to the filter plate. On completion of this step, thefilter plate was immediately stacked onto an empty 384well PP plate (elution plate), and the combined plates wereloaded into the centrifuge and spun at 800 rcf for 2 min.Subsequent to the separation step, the filter plate wasrestored, and the elution plate containing the eluted sampleswas transported to a CyBio FeliX liquid-handling system(Analytik Jena). There, 3 µL of every well was transferredfrom the 384-well elution plate to one quadrant (Q1/Q2/Q3/Q4) of a 1536-well plate. After four consecutive transfersfrom four individual elution plates (elution plate 1 Q1,elution plate 2 Q2, etc.), 3 µL of a mixture containinginternal standard (500 nM final concentration) and the standard for the SEC quality control (QCref; 250 nM final concentration) in acetonitrile were added to every well of the1536-well plate using a Certus Flex Micro Dispenser(Gyger, Gwatt, Switzerland). Plates were then sealed andstored until preparation of the MALDI target plates. A flowchart of the automated binding assay and a Gantt chart ofthe process sequence are provided in the online supportinginformation (Suppl. Fig. S2A and Suppl. Fig. S2B).Hit Confirmation WorkflowProcess steps and plate handling were executed as describedabove. For hit confirmation experiments, 50 nL of 5 µg/µLor 10 mM compound solutions were transferred to the wellsof two individual 384-well PP assay plates (sample plateand low control plate). A third plate received 50 nL DMSOper cavity (high control plate). Sample plates and high control plates were further processed by dispensing 5 µL of0.4 mg/mL PTP1B CA in assay buffer into every well ofthe plates, while low control plates received 5 µL BGG(0.4 mg/mL) instead. After incubation, mixtures were transferred to assay-ready filter plates using automated liquidhandling. Filter plates containing samples or low controlswere then stacked onto 384-well PP plates containing 50 nLDMSO per cavity, while high control–containing filter

Simon et al.plates were stacked onto plates containing 50 nL of 10 diluted (0.5 µg/µL or 1 mM) compound solutions in DMSO.Plates were then centrifuged, and eluates were subsequentlytransferred to quadrants of a 1536-well microtiter plate(samples Q1, low controls Q2, and high controls Q3). Q4 received 3 µL of 10 mM NH4Ac buffer (pH 7.3) asnegative control. Addition of standards and further plateprocessing were conducted as described above.MALDI Target Preparation4-HCCA was dissolved at 3 mg/mL in a 1:1 mixture of acetonitrile and 5 mM NH4H2PO4/0.1% TFA (v/v) with the aidof vigorous vortexing. The CyBio Well vario liquid-handling system (Analytik Jena) equipped with ceramic tipsand operated in 1536-well format was used to conduct drieddroplet spotting, providing highly homogeneous spotshapes. Here, assay plates were centrifuged at 1000 rpm for60 s, and the seals were removed before 100 nL matrix solution and 100 nL sample were aspirated successively fromthe matrix reservoir and the assay plate, respectively, anddispensed together onto the plain steel MALDI target plates.The target plates were dried in a vacuum chamber beforethe spotting process was repeated. Between the first andsecond spotting steps and after the second spotting step, theceramic tips were washed three times with isopropanol/0.1% aqueous TFA (70/30, v/v). Following successfultransfer and drying of matrix–analyte mixtures, an on-targetwashing procedure was applied using 10 mM NH4H2PO4/0.3% TFA. Processed MALDI target plates were storeduntil analyzed. After every preparation cycle, three repetitive washing cycles using 0.1 M NH4OH followed by threecycles of 70% isopropanol and 30% water/0.1% TFA (v/v)were carried out on the liquid-handling system to preventcarryover and clogging of the tips.MALDI-TOF-Based HTS ReadoutMass spectra were acquired with a rapifleX MALDI-TOF/TOF instrument from Bruker Daltonics, including aSmartbeam 3D laser. FlexControl (v. 4.0), flexAnalysis(v. 4.0), and rapifleX MALDI PharmaPulse (MPP; v. 2.2),all from Bruker Daltonics, were used for MS acquisitionand data analysis. Target plates were loaded onto an OrbitorRS (Thermo Fisher Scientific) robotic system controlled bythe laboratory automation software Momentum (v. 4.2.3,Thermo Fisher Scientific) and automatically inserted intothe MALDI-TOF device. Mass spectra were acquired in themass range of m/z 200–1100 to cover target analyte, internal standard, and controls of the binding assay. Therefore,2500 laser shots per sample spot were accumulated in20-shot increments across a small spiral spot raster in positive or negative ionization mode using a 160 ns pulsed ionextraction with a 10 kHz laser frequency, a digitizer setting47of 5.00 GS/s, and an M5 defocus Smartbeam parameter at a50 50 µm scan range, which resulted in a 124 124 µmfield size. The laser power was adjusted manually beforeevery start of a batch process to reach a sufficient signalintensity for the internal standard ( 2 105 cts in positiveionization mode; 5 104 cts in negative ionization mode).The acquired spectra were processed in MPP2.2 SynthesisScreening mode or using flexAnalysis with a centroid peakdetection set to a signal-to-noise ratio (S/N) 15 and apeak width of m/z 0.05.Data AnalysisFor data evaluation, signal area values [area under the curve(AUC)] extracted from flexAnalysis result files were referenced against corresponding standard signals for small molecules [internal standard (IS)] and quality controls (QCref),and reported as ratio values. When spectra were processedby MPP2.2 Synthesis Screening mode, ratio values fordetected compound signals and QC signals were extractedstraight from result files. In all cases, signals measured withan m/z value deviating more than 0.032 from the calculatedtheoretical value were rejected and not used for further calculations. The ratio of QC peptide signal and QC standardsignal had to stay lower than the threshold of 1.0 (representative for 1% SEC leakage) for measured samples to beaccountable for further analysis. For signals within the massdeviation threshold, the proportional increase of the signalratio in sample wells compared to the mean of negativecontrol–containing wells was calculated and expressed asFold Change (FC) values, according to the followingequation:Fold Change ( FC ) Ratio Cpdmean Ratio Negative control(1) Small-molecule analyte signals reaching FC values of 2.5were considered as hits in the screening workflow or incompound detectability analysis. In cases in which signalsat compound m/z were observable in sample measurementsbut not in negative controls, the FC value calculation wasomitted, and the compound was counted as an assay hit.Measurements failing the QC threshold or not containingany measurable IS were reported together with compoundinformation and aggregated in a dropout list to enableretesting in a separate assay run.Data from hit confirmation workflow experiments wereanalyzed in a similar fashion as described for screeningworkflow data. Ratio values were calculated for compoundand QC signals from Exp. A (sample), Exp. B (low control),and Exp. C (high control) measurements. In addition, a FCvalue was calculated from high control compound signalsand the mean of observable negative control ratio values.

48SLAS Discovery 26(1)Relative binding affinities (RBAs) for specific compound–protein interactions were calculated via the followingequation:RBA [ % ] Ratio Cpd [ Exp. A] Ratio Cpd [ Exp.B ]Ratio Cpd [ Exp.C ](2) To qualify as a hit in the hit confirmation workflow, testcompound data had to fulfill the following criteria: m/zdeviation 0.032, internal standard detected in all threeexperiments (A–C), QC passed (QC ratio 1.0) in all threeexperiments, FC 2.5, and RBA 5%. Data were furtherprocessed using GraphPad Prism (v. 8.30; GraphPadSoftware, La Jolla, CA). A more detailed description ondata handling and processing is provided online in the supporting information. The assignment of compounds to thecorresponding measurements was achieved by softwareaided deconvolution of every 1536-well assay plate to thecorresponding 384-well substance plates in the course ofdata analysis, as we have described in Ref. 21.Further MethodsA summary table compiling information on components andcontrols of the presented MALDI-TOF AS-MS concept targeting PTP1B (Suppl. Table S1) and method informationon the MALDI-TOF MS calibration procedure, the loadingof SEC resin to 384-well filter plates, AlphaScreen experiments, differential scanning fluorimetry experiments, theMALDI-TOF-based PTP1B activity assay, isothermal titration calorimetry experiments, NMR spectroscopy, and further data handling and processing can be found in theSupplemental Methods section of the Supplemantal Material.Results and DiscussionMALDI-TOF AS-MS WorkflowsSEC-based separation performed directly in microtiterplates is a straightforward and effective approach that isamenable to automation on current screening platforms. Toenable screening of small-molecule libraries for noncovalent binding to a protein target, two workflows weredesigned based on an in-plate SEC-enabled separation procedure in 384-well format. A schematic depiction of bothworkflows is provided in Figure 1. In the first workflow(Screening workflow), individual incubations of protein andtest compounds are transferred to SEC resin-filled 384-wellfilter plates, and the components are separated according tothe size exclusion cutoff on centrifugation. Compoundsinteracting with the target pass the SEC resin and can bedetected in subsequent analysis. To control for efficientcutoff separation, a quality control (cutoff QC) is added tothe assay buffer at a fixed concentration, which allows forquantification of SEC leakage. To account for intrinsicMALDI-TOF signal variabilities, two reference standardsare added to every sample after elution from the filter plate.While the IS is referenced against compound signals(Ratiosample), the quality control standard (QCref) definesthe threshold for acceptable SEC leakage and is used to precisely quantify the amount of QC in eluted sample fractions. Compounds detected at levels higher than certainthresholds in sample wells compared to DMSO controlwells are considered a screening workflow hit. Because thisfirst workflow provides only a qualitative measure of protein binding and does not account for unspecific interactions, a second assay (Hit confirmation workflow) includingcontrols for every test compound was implemented. Here,in addition to the regular sample incubation of test compound and protein target, a low control that detects unspecific binding and a high control that resembles the maximumachievable compound concentration after elution areincluded. While for low controls the protein of interest issubstituted for an unrelated protein or an unfavored proteinisotype, the high control is performed with the target but inthe absence of test compound. After elution from the filterplate, dilutions of test compounds are spiked into high controls at concentrations that mimic full 1:1 target saturation.After analysis, controls are used to calculate the specificRBA, which is a quantitative measure of specific proteinbinding. Combining the two presented workflows conceptually enables screening of small-molecule compoundlibraries for qualitative target binding followed by stringent hit verification and relative quantification of ligandaffinity of primary screening hits using a label-freeMALDI-TOF MS readout. Following workflow design, weevaluated critical method parameters for the SEC-based inplate separation process and for MALDI-TOF MS-enabledsmall-molecule detection.SEC-Based In-Plate Separation ProcessEfficient retention of unbound ligand and buffer components is a prerequisite for robust data generation by the twopresented workflows. Optimization of SEC separation efficiency was carried out with buffer containing fluorescein asa tracer. A bed volume of 65 µL SEC resin per well caused 99.5% retention of the fluorescent tracer and was used forfurther experiments (Suppl. Fig. S1A). To consistentlytrack the separation capacity of the assay, a control peptide(QC; ETDYYRKG-amide; M 1029.458 Da) and its reference standard (QCref; ETDYYR[K]G-amide; M 1039.488 Da) with mass values well outside the typicalmass window for small-molecule analytes were selected.Linearity of QC concentration-dependent signal response

Simon et al.49Figure 1. Scheme of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) affinity selection mass spectrometry(AS-MS) assay workflows. (A) Screening workflow: Individual incubation solutions are transferred from 384-well assay plates ontosize exclusion chromatography (SEC) resin-loaded 384-well filter plates. Protein and protein–ligand complexes are separated fromunbound components via centrifugation, and eluates are captured. After addition of standards, samples are spotted onto MALDItarget plates and analyzed by MALDI-TOF MS. Compounds binding to the protein target (Case 1) produces mass specific signals in MSanalysis compared to samples in which the compound is retained by the SEC resin (Case 2). Intactness of the SEC-based separationprocedure is monitored by quantifying a quality control (QC) peptide to detect false positives caused by inefficient separation (Case3). (B) Hit confirmation workflow: In addition to regular sample incubation (Exp. A), controls containing either an unrelated target(Exp. B) or pure DMSO instead of test compound (Exp. C) are prepared. In the case of high controls, compound is spiked to elutedhigh controls to simulate full target binding. The amount of protein binding for samples (Exp. A) is then quantified in relation to lowand high controls, respectively.times of 74 min per 1536-well plate, which translated into atheoretical throughput capacity of 29,890 samples per dayper MALDI instrument (19 1536-well plates, 76 assayplates/filter plates, and 26,700 test compounds; 2.9 s/sample; Suppl. Fig. S2B). We would like to note that additionalprocess optimization, parallelization of process steps, andthe introduction of filter plates in higher-density formattogether hold the potential for further increasing the dailythroughput of the platform. Here, we demonstrate that HTScapable sample preparation by automated in-plate SEC isachievable on a standard automation platform withoutmajor modifications.Optimization of MALDI Matrix Compositionand Deposition for Small-Molecule DetectionInterferences in the low mass region arising from background signals caused by the matrix and chemotypedependent low ionization efficiency have been major issuesin MALDI-TOF MS analysis of small molecules. A multitude of approaches to minimize background interferencewhile maximizing compound ionization have been reported,suggesting a variety of matrix constituents, additives, solidsupports, and preparation procedures.22–26 Many of theseimprovements, however, are valid only for specific analytechemotypes, require special treatment of MALDI targetplates, or interfere with automation processes. Therefore, aset of 10 different MALDI matrices, which have proven tobe compatible with the applied automated spotting procedure, were evaluated for suitability. Co-crystallization withα-cyano-4-hydroxycinnamic acid (HCCA) producedacceptable S/N values for each of eight chemically diversesmall molecules and the QC peptide (1 µM in demineralized H2O) in positive and/or negative ionization mode(Suppl. Fig. S3A). Further optimization showed that bestresults were obtained using a solution of 3 mg/mL HCCA inacetonitrile/H2O 1:1 containing 10 mM NH4H2PO4 0.3%trifluoroacetic acid and applying a double dried dropletspotting procedure followed by an on-target washing step(Suppl. Figs. S3B and S4). Because HCCA producesintense mass signals around m/z 190 (corresponding to[M H] and [M-H] of the HCCA molecule), measurements were focused on a mass range of m/z 200–1100.Figure 2B depicts representative mass spectra recorded in

50SLAS Discovery 26(1)Figure 2. Evaluation of in-plate size exclusion chromatography (SEC) and matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometry (MS) small-molecule detection. (A) Retention profile of eluates from a 384-well filter plate filledwith 65 µL SEC bed volume per cavity and challenged with 10 µL of 1 µM fluorescein solution or 100 µM quality control (QC)peptide, respectively. (B) Example spectra of a seven-component mixture of five small molecules (C01–C05) and the standards IS(internal standard) and QCref (the SEC quality control) (500 nM each). Blank spectra of pure co-crystallization matrix (black) areoverlaid with sample spectra (red).

1536-well format were performed on a fully automated sys-tem featuring a modular design and robotic plate transporta-tion. At the beginning of each iterative cycle, a 384-well compound plate was transported to a CyBio Well vario liq-uid-handling system (Analytik Jena, Jena, Germany) equipped with a 384-well format capillary head to transfer