Efficient Screening of Combinatorial Peptide Libraries by Spatially Ordered Beads Immobilized on Conventional Glass Slides

Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput “all-on-one chip” system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.


Resin Preparation
TentaGel HL NH2 resin was swollen in DMF for 1 h and washed with DMF. HMBA (0.1 mol/L, 4 eq), HCTU (7.2 eq) and NMM (16 eq) were dissolved in DMF and added to the resin for 1 h. The HMBA coupling was repeated using identical amounts of each reagent. The resin was washed and subjected to the Kaiser test to confirm that the reaction had gone to completion [1].
For the incorporation of the first amino acid, Gly-Fmoc (3 mol/L, 10 eq) and DIC (5 eq) were dissolved in DCM and stirred at 2°C for 20 min. The mixture was added to the resin, followed by dropwise addition of DMAP (0.1 eq, 50 mmol/l in DMF). After 10 h, the resin was washed with DMF and DCM. Coupling was repeated once overnight. Fmoc was cleaved off by 20% piperidine in DMF for 30 min. The coupling efficiency was determined by screening of the deprotection solution. This was performed using an Evolution 220 UV-Visible Spectrophotometer by Thermo Fisher Scientific (Massachusetts, USA), recording the specific absorption of the dibenzofulvene-piperidine adduct at 301 nm. The yield was about 60 %.

Peptide Synthesis
The spacer sequence GGTERSG for the peptide libraries, test peptides (DYKDDDDK, DTHFPIGG, VPFHTDGG, KFVPFKKS) and resynthesized peptides from screening (GRIHGPR, GIHPFGR, GEPFSIP, SHSGIFR) were manually synthesized by adding Fmoc-amino acids (0.1 mol/L, 4 eq.) in DMF to the resin using HCTU (4 eq.), and NMM (8 eq.) for 1 h. Fmoc was cleaved off by 20% piperidine in DMF for 30 min. Beads were washed thoroughly with DMF after each step. Fmoc monitoring of the deprotection solution was performed by UV/VIS spectrometer. Finally, the resin was treated with a mixture of 95 % TFA, 2.5 % TIS, 2.5 % lab water to remove side chain protecting groups, followed by intensive washing with DCM and drying of the resin in vacuo.

Synthesis of Peptide Library 1
The library was synthesized using 20 mg of a TentaGel HL NH2 resin modified with HMBA and the Spacer sequence GGTERSG. The variable peptide segment/peptide library was synthesized manually by splitting the resin into eight equal aliquots. Coupling of each aliquot was performed using 8 eq. of each Fmoc protected amino acid (Thr, Ile, Asp, Lys, Tyr) and 0.4 eq of the corresponding Boc-protected amino acid in DMF with HCTU (4 eq.), and NMM (8 eq.) for 1 h or overnight. All aliquots were mixed for washing and Fmoc deprotection. This cycle of splitting and mixing was repeated eight times. Finally, the resin was treated with a mixture of 95 % TFA, 2.5 % TIS, 2.5 % lab water to remove side chain protecting groups and Boc protecting groups from the truncated peptide sequences, followed by intensive washing with DCM and drying of the resin in vacuo.

Synthesis of Peptide Library 2
The library was synthesized using 500 mg of a TentaGel HL NH2 resin modified with HMBA and the spacer sequence GGTERSG. The variable peptide segment/peptide library was performed as described before using 8 eq. of the respective Fmoc-protected amino acids (Gly, His, Ile, Ser, Phe, Pro, Glu, Arg) and 0.4 eq (5 %) of the corresponding Boc-protected amino acids.

Chip Preparation
A plain microscope slide (J. Melvin Freed) with a size of 75 × 25 mm 2 was modified by attachment of the 3M XYZ-Axis electrically conductive double-sided adhesive Tape 9713, which is an isotropically conductive, pressure sensitive tape. The tape conducts electrically through the thickness (Z-axis) and in the plane of the adhesive (X, Y planes) and is ideal for MALDI-TOF MS/MS measurements. It contains a nickel-plated carbon scrim. The modified microscope slide was fixed temporarily in a custom-made slide holder. A sieve made from stainless steel with a mesh of 100 μm was attached on the surface of the adhesive tape. The dried beads were spread on the sieve and gently dispersed to allow the resin to fill the meshes. Slight pressing on top of the beads ensured attachment on the sticky surface. Figure S1. of a layer of 75 μm TentaGel beads, immobilized on a surface of a microscope slide modified with an electrically conductive, double-sided adhesive tape. Small nickel wires are visible in the background, responsible for the electrical conductivity.

Cleavage of Peptides and Matrix-application via Airbrush Gun
The incubation was followed by regeneration of the chip. To remove all salts, the chip was extensively washed with lab water and dried. For peptide cleavage from the bead surface, the chip was placed in a chamber with 2 ml of aqueous ammonia solution (30%) at the bottom. The linker was efficiently cleaved within 2 h. Matrix application was achieved using a conventional Agora-Tec airbrush (Airbrush Pistole Kit AT-AK-01, Agora-Tec, Schmalkalden, Germany). 2 mL of matrix solution was used per chip, consisting of 20 mg DHAP in ethanol, dibasic ammonium citrate (18 mg/mL in lab water) (3:1 v/v). The chip was placed 15 cm under the tip of the airbrush and a constant pressure of 2 bar was applied to the airbrush gun. This ensured the formation of a thin matrix layer on the surface of the bead ( Figure S2).

Sequencing of Test Peptides by MALDI-TOF MS/MS (fragmentation)
For MS/MS experiments, beads of the four test peptides (DYKDDDDKGGTERSGG, DTHFPIGGGGTERSGG, VPFHTDGGGGTERSGG, KFVPFKKSGGTERSGG) were immobilized on a chip, peptides were cleaved from the beads and matrix was applied as previously described. Sequencing was performed using a Bruker autoflex II smartbeam (Bruker Daltonics GmbH, Leipzig, Germany) with a MALDI laser source (355 nm, 200 Hz) and a TOF detector. MS spectra were gained in positive reflectron mode with an average of 500 laser shots directly on the resin bead. The precursor ions from three beads of each peptide were selected by taking the most intense peaks, corresponding to the theoretical mass of peptides. Fragmentation spectra were recorded using on average 5500 laser shots, using a precursor mass window of m/z = ± 10. Argon was used as collision gas. Data processing and database search were performed by using a custom-made peptide library search tool (Seq2SpecPeptides), automatically identifying the corresponding peptide sequences. Ion scores were introduced to rank the goodness of identification. The software is based on a code [2] used for the sequence identification of PEG-block-Tla/Michael-oligomers [3]. The results are shown in Tables S1-S4.  DTHFPIGGGGTERSGG, VPFHTDGGGGTERSGG, KFVPFKKSGGTERSGG) were immobilized on a chip, the peptides were cleaved from the beads and matrix was applied as previously described. MS spectra were obtained in positive reflectron mode with an average of 1500 laser shots directly on the resin bead. The obtained ladder-sequence spectra were processed by using a custom-made peptide library search tool (Seq2SpecPeptides), automatically identifying the corresponding peptide sequences. The peptide sequences are sorted by the number of the recognized truncated peptide sequences. The results are shown in Tables S5-S8.

Incubation with Anti-FLAG Antibody M2
The chip with the immobilized peptide library 1 was pre-swollen in PBS-T BSA (pH 7.5; 0,1 % Tween 20; 1 % BSA). For prescreening, the chip was incubated with anti-Mouse IgG-Atto 633 antibody diluted 1:10,000 in PBS-T BSA for 1 h to identify false-positive beads. After incubation, the slide was removed from the solution, washed three times with lab water and directly transferred to the fluorescence scanner. Fluorescence scanning was performed using a Microarray Scanner MArS (Ditabis AG, Pforzheim, Germany) with a laser wavelength of 532 nm to locate the beads by autofluorescence of the beads and 635 nm for detecting bound, labeled antibody. The PMT gain was set to 65% for 532 nm and 70% for 635 nm and the resolution to 10 μm. For regeneration, the chip was gently shaken for 30 min in an aqueous 6 M guanidine hydrochloride solution, 5 min in lab water and washed three times with PBS-T BSA). Incubation with monoclonal Anti-FLAG M2 antibody diluted 1:10,000 in PBS-T BSA for 15 h and washed five times with PBS-T BSA. Washing was followed by incubation with the anti-Mouse IgG-Atto 633 antibody diluted 1:10,000 in PBS-T BSA for 1 h. Fluorescence scanning was performed as previously described. 33 beads were selected for peptide sequencing, which showed at least three times the fluorescence intensity compared to the prescreening scan ( Figure S3).

Ladder Sequencing of the Peptides Selected from Library 1 after Anti-FLAG Antibody Incubation
Coordinates obtained from the incubation experiments were transformed into coordinates compatible with the Bruker software. This was achieved by selecting four beads on the corners of the chip in the fluorescence scan and correlating the coordinates by linear regression in an excel sheet in the MALDI software. MS spectra were gained in positive reflectron mode with an average of 1500 laser shots directly on the resin bead. The obtained ladder sequence spectra were processed by using a custom-made peptide library search tool (Seq2Spec, see ESI), automatically identifying the corresponding peptide sequences. The peptide sequences are shown in Table S9.

Ladder Sequencing of the Peptides Selected from Library 2 after Polyclonal Mouse IgG Incubation
The chip with the immobilized peptide library 2 was pre-swollen in PBS-T BSA. Prescreening was performed with the anti-Mouse IgG-Atto 633 antibody as previously described. After regeneration, the chip was incubated with polyclonal mouse IgG diluted 1:10,000 in PBS-T BSA for 15 h, washed five times with PBS-T BSA and fluorescence of the beads was detected as in the anti-FLAG incubation. Fluorescence scanning was performed as previously described. Nine beads were selected for peptide sequencing, which showed at least a three times higher fluorescence intensity compared to the prescreening ( Figure S4). Green autofluorescence is used to locate all TentaGel beads and red fluorescence was used to identify beads bound by the polyclonal mouse IgG.

Peptide Ladder-sequencing by MALDI TOF MS/MS of Library 2
Coordinates were transformed, and binding peptides were sequenced as previously described.
S H S G I F R Color code: Blue = basic AA, red = acidic AA, yellow = hydrophobic AA, orange = aromatic AA, green = polar AA.

SPR Chip Preparation
Surface Plasmon Resonance (SPR) experiments were performed by a Reichert SR7500DC dual channel SPR system (Reichert, USA). As a flow buffer, HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Tween 20) was used throughout all experiments. Immobilization of the antibody was performed on a gold biosensor chip HC200 (XanTec bioanalytics, Germany) using the following amine coupling protocol: Coupling buffer was prepared by dissolving EDC (0.1 M) in MES/NHS buffer (50 mM MES pH 6.0, 0.1 M NHS) and directly injected over both the sample and reference channel for 4 min (20 μl/min). 10 μg of the antibody was diluted in 100 μl of 1 mM sodium acetate (pH 5.0) and injected over the sample channel for 1.5 min (50 μl/min). For quenching, 1 M ethanolamine in lab water was injected over both, the sample and reference channel for 1 min (20 μl/min).

SPR Characterization of Anti-flag Binding Peptides
Two peptides identified in the anti-FLAG antibody screening, the original FLAG-peptide and a peptide created as a consensus peptide by choosing the most abundant amino acid at every position, were synthesized by peptides & elephants GmbH (Hennigsdorf, Germany). For the determination of their affinity, an SPR chip was prepared as previously described using the monoclonal anti-FLAG antibody, clone M2. 1 μM of the peptides was dissolved in 1 mL HBS-EP buffer. The stock solution was diluted resulting in the following concentrations: 6 nM, 18 nM, 55 nM, 166 nM, 500 nM and 1000 nM. The peptides affinities were measured by injecting the dilutions over both the sample and reference sides for 3.5 min (20 μl/min). The signal measured on the reference side was subtracted from the signal measured on the sample side. For determination of the affinity constant, the signal intensity of the equilibriums state was plotted against the concentration of peptide injected. The calculation was done by the TraceDrawer Data Analysis Software. The results are shown in Figure  S5.   Figure S5. binding results for FLAG (DYKDYDKD), peptide 1 (DYKDYDKD) a FLAG epitopecontaining sequence, peptide 2 (DYIDYDYK) a mimotope-containing sequence and peptide 3 (DYDIYIYD) representing a consensus sequence of the most abundant amino acids at each position.
The FLAG antibody was immobilized on an HC200 chip and peptide solutions in different concentration were injected. The intensity of the signal in equilibrium is plotted against the concentration.

SPR Characterization of Mouse IgG Binding Peptides
Four peptides identified in the mouse IgG screening were resynthesized using standard Fmoc peptide synthesis. For the determination of the affinity, an SPR chip was prepared as previously described using the mouse IgG. 10 μM of the respective peptide was dissolved in 1 mL HBS-EP buffer. The stock solution was diluted, resulting in the following concentrations: 1 μM, 5 μM, 10 μM, 50 μM, 100 μM. The peptide affinities were measured by injecting the dilutions over both the sample and reference sides for 5 min (10 μl/min). For the determination of the affinity constant, the signal intensity of the equilibrium state was plotted against the concentration of peptide injected. The calculation was done by the TraceDrawer Data Analysis Software. The results are shown in Figure  S6.

Monitoring of Dibenzofulvene-Piperidine Adducts in Ladder Synthesis
The Fmoc-protecting group was cleaved off by 20% piperidine in DMF for 30 min. Fmocscreening of the deprotection solution was performed by recording the specific absorption of the dibenzofulvene-piperidine adduct at 300 nm. Dibenzofulvene-piperidine adduct monitoring of two test peptides (DTHFPI-Linker, VPFHTD-Linker) are shown in Figure S7.
Due to inconsistent dibenzofulvene-piperidine adduct formation in the split and mix procedure, a Fmoc-adduct screening during the library synthesis was not performed.