Ultra-large chemical libraries for the discovery of high-affinity peptide binders

High-diversity genetically-encoded combinatorial libraries (108−1013 members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only ~ 106 compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 108 members—a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 106–108. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3–19 nM-affinity, α/β-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of β-amino acids in facilitating a key binding contact.


Supplementary Note 1: Theoretical diversity vs. sampling diversity
Given the large theoretical diversity of this library relative to the diversity sampled (limited by the number of beads used in split-and-pool synthesis), it is unlikely the full HA epitope would be rediscovered. There is a 0.001% chance of finding a given 9-mer sequence in a library comprising 2 x 10 6 random 9-mers, with 18 possible monomers at each position (theoretical diversity = 2 x 10 11 ).
For automated syntheses: syntheses were carried out at 90 °C. Amide bond formation was effected in 8 s, and Fmoc removal was carried out in 8 s with 20% (v/v) piperidine in DMF. Individual cycle times were each about 40 s.
For manual flow-based syntheses: reagents and solvents were delivered to a stainless steel reactor, which contained the resin, by either an HPLC pump (DMF or 20% (v/v) piperidine in DMF) or a syringe pump (active esters of Fmoc-amino acids). The reactor was submerged in a water bath for the duration of the synthesis and the temperature was maintained at 70 °C. The procedure for each coupling cycle included: a 30 second coupling with a mixture of Fmoc-protected amino acid (1 mmol), HBTU (0.95 mmol), and diisopropylethyl amine (DIEA; 2.9 mmol, 500 μL) in 2.5 mL of DMF, at a flow rate of 6 mL/min (for the coupling of tryptophan and histidine, 190 μL of DIEA was used to minimize racemization); 1 min DMF wash, at a flow rate of 20 mL/min; 20 second deprotection with 20% (v/v) piperidine in DMF, at a flow rate of 20 mL/min; and 1 minute DMF wash, at a flow rate of 20 mL/min.
After each synthesis was complete, resins were washed with DCM (5x) and dried under reduced pressure.
Global side chain deprotection and cleavage from solid support were carried out by treatment of dry resin with a solution of 94% (v/v) TFA, 2.5% (v/v) ethanedithiol, 2.5% (v/v) water, and 1.0% (v/v) triisopropylsilane, for 2 h at ambient temperature (~1.5 mL of deprotection solution/50 mg of resin). TFA was then evaporated under a stream of nitrogen, and crude peptide was precipitated by addition of cold diethyl ether. Precipitated peptide was triturated (3x) with cold diethyl ether, dissolved in 50/50 water/acetonitrile (0.1% TFA), passed through a 0.2 μm PTFE syringe filter, and lyophilized.
Crude peptides were purified by semipreparative reverse phase HPLC, using an Agilent mass directed purification system (1260 infinity LC and 6130 single quad MS). For a typical purification, peptides were dissolved in 95/5 water/acetonitrile (0.1% TFA) and passed through a 0.2 μm PTFE syringe filter. The resulting peptide solution was then loaded onto a 9.4 x 250 mm column (Agilent Zorbax 300SB-C3; 5 μm particle size; 300 Å pore size) and purified using a linear gradient of 1 to 61% acetonitrile (0.1% TFA) over 60 min (4 mL/min flow rate). Fractions containing the desired product were pooled and an aliquot taken for LC-MS analysis. The remainder was lyophilized.
For LC-MS characterization: LC-MS data were acquired using an Agilent 6550 quadrupole time-of-flight LC-MS. Samples were run on an Agilent Zorbac 200SB-C3 column (2.1 x 150 mm, 5 μm particle size, 300 Å pore size). Total ion current (TIC) chromatograms were plotted, and mass spectra were integrated over the principal TIC peak, shown below.

Preparation of biotinylated 12ca5:
Biotin-(PEG)4-NHS ester (2.0 mg, 3.3 μmol) was weighed into a plastic tube and dissolved in 1.06 mL of DMF ([Biotin-(PEG)4-NHS] = 3.3 mM). Anti-HA antibody (4.71 mg/mL in 1x PBS, 1.06 mL, 33 nmol) was transferred to a plastic tube and to this was added 123 μL of 1M sodium bicarbonate, pH = 8.0. Biotin-(PEG)4-NHS ester (3.3 mM in DMF, 53 μL, 170 nmol) was added dropwise to solution of anti-HA antibody, and reaction was placed on a nutating mixer for 2 h at ambient temperature. Reaction was quenched with addition of 20 mM Tris, 150 mM NaCl, pH = 7.5 (4 mL). Mixture was then filtered through a 0.2 μm PTFE syringe filter, and purified by FPLC (ÄKTA Prime Plus Liquid Chromatography System, GE Healthcare). Concentration of biotinylated 12ca5 was measured by absorption at 280 nm, using a determined extinction coefficient of 2.0 x 10 5 M -1 cm -1 . Protein was stored at 4 °C and not subjected to freeze-thaw cycles.

Affinity capture of 12ca5-binding peptides-effect of ligand concentration:
Preparation of 12ca5-functionalized magnetic beads: 100 μL portions of MyOne Streptavidin T1 Dynabeads (10 mg/mL; 1 mg; 0.13 nmol IgG binding capacity) were transferred to 1.7 mL plastic centrifuge tubes, and placed in a magnetic separation rack (New England Biolabs, cat# S1506S). The beads were washed 3 times with 1 mg/mL BSA, 0.02% Tween 20, 1x PBS, and then treated with 100 μL portions of biotinylated 12ca5 (1.5 μM; 0.15 nmol). The resulting suspensions were transferred to a rotating vertical mixer, and kept for 15 min at ambient temperature. After this time, the beads were returned to the separating rack, the supernatant was removed, and the beads were washed 4 times with 1 mL each of 1 mg/mL BSA, 0.02% Tween 20, 1x PBS.
Affinity capture: 1 mL solutions containing 1 mg/mL BSA, 0.02% Tween 20, 1x PBS, and either 1 nM/peptide (1 pmol) or 10 pM/peptide (10 fmol) 12ca5 control binders (Supplementary Table 1) were prepared in 1.7 mL plastic centrifuge tubes, and chilled on ice for 10 min (the 12ca5 binders were added from mixtures containing 1 µM/peptide or 10 nM/peptide in 6 M guanidine hydrochloride, 200 mM phosphate, pH 7 buffer). The resulting chilled solutions were then added to 1 mg portions of 12ca5-functionalized magnetic beads, and the resulting suspensions were kept on a rotating vertical mixer (1 h, in 4 °C cold room).

Elution:
The centrifuge tubes containing the bead suspensions were transferred to the magnetic separation rack. The beads were isolated, and washed 3 times with 1 mL each of chilled 1x PBS (beads were exposed to buffer for a total of ~6 min). Then, each drained bead aliquot was treated with 2 x 150 µL of 'elution buffer' (6 M guanidine hydrochloride, 200 mM phosphate, pH 7.0 buffer containing 1 fmol/µL Peptide Retention Time Calibration Standard (PRTC; Pierce, cat# 88320; for use as an internal reference in MS-based quantitation)).
Preparation of 'reference' samples: 1 pmol and 10 fmol/peptide 'reference' samples were prepared by dilution of 1 µM/peptide or 10 nM/peptide 12ca5 binder mixture stock solutions (in 6 M guanidine hydrochloride, 200 mM phosphate, pH 7 buffer) into 300 µL of 'elution buffer'. These samples contained the amount of peptide that would be present in elution, if 100% of the peptide were retained by affinity capture. NanoLC-MS: 5 µL portions of the combined eluates were analyzed by nanoLC-MS, alongside 5 µL portions of 'reference' samples (16.7 fmol/peptide or 167 amol/peptide loading for '1 nM' or '10 pM' conditions, respectively). Analysis was performed on an EASY-nLC 1200 (Thermo Fisher Scientific) nano-liquid chromatography handling system connected to an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific). Samples were run on a PepMap RSLC C18 column (2 μm particle size, 15 cm x 50 μm ID; Thermo Fisher Scientific, P/N ES801). A nanoViper Trap Column (C18, 3 μm particle size, 100 Å pore size, 20 mm x 75 μm ID; Thermo Fisher Scientific, P/N 164946) was used for desalting. The standard nano-LC method was run at 40 °C and a flow rate of 300 nL/min with the following gradient: 1% solvent B in solvent A ramping linearly to 61% B in A over 40 or 60 min, where solvent A = water (0.1% FA), and solvent B = 80% acetonitrile, 20% water (0.1% FA). Positive ion spray voltage was set to 2200 V. Orbitrap detection was used for primary MS, with the following parameters: resolution = 120,000; quadrupole isolation; scan range = 200-1400 m/z; RF lens = 30%; AGC target = 1 x 10 6 ; maximum injection time = 100 ms; 1 microsan.

Generation of dose-response curve:
A dose-response curve was generated by analyzing 5 µL portions of the 'reference samples', containing 16.7 fmol/peptide, 1.67 fmol/peptide, or 167 amol/peptide. MS detector counts for each peptide were determined from the apex of extracted ion current chromatograms, and plotted vs. sample loading to verify the linearity of response over the sample loading range of interest.
Quantitation of sample recovery: MS detector counts for each peptide were determined from the apex of extracted ion current chromatograms. Recoveries were taken as the ratio of counts for samples obtained by affinity selection vs. the 'reference' samples. To account for run-to-run variability, these ratios were adjusted based on the counts obtained for internal standard (Peptide Retention Time Calibration Standard).

Affinity capture of 12ca5-binding peptides-effect of capture protocol:
'Direct capture' with 12ca5-functionalized magnetic beads: Preparation of 12ca5-functionalized magnetic beads was carried out as in Affinity capture of 12ca5-binding peptides-effect of ligand concentration. Affinity capture treatments were performed as in Affinity capture of 12ca5-binding peptides-effect of ligand concentration, from 1 mL volumes of 1 nM, 100 pM, or 10 pM/peptide mixtures of 12ca5-binding peptides (Supplementary Table 1).
Elution, nanoLC-MS analyses of elution and 'reference' samples, and quantitation were performed as in Affinity capture of 12ca5-binding peptides-effect of ligand concentration.

Affinity capture of 12ca5-binding peptides-effect of magnetic bead concentration:
'Direct capture' with 12ca5-functionalized magnetic beads: 100 μL (1 mg; 0.13 nmol IgG binding capacity) or 1 mL (10 mg; 1.3 nmol IgG binding capacity) portions of MyOne Streptavidin T1 Dynabeads (10 mg/mL); were transferred to 1.7 mL plastic centrifuge tubes, and placed in a magnetic separation rack. The beads were washed 3 times with 1 mg/mL BSA, 0.02% Tween 20, 1x PBS, and then treated with 100 μL or 1 mL portions of biotinylated 12ca5 (1.5 μM; 0.15 nmol or 1.5 nmol). The resulting suspensions were transferred to a rotating vertical mixer, and kept for 15 min at ambient temperature. After this time, the beads were returned to the separating rack, the supernatant was removed, and the beads were washed 4 x 1 mL each with 1 mg/mL BSA, 0.02% Tween 20, 1x PBS.
Affinity capture was performed from 1 mL solutions containing 1 mg/mL BSA, 0.02% Tween 20, 1x PBS, and 1 nM/peptide (1 pmol) 12ca5 control binders (Supplementary Table 1). Elution, nanoLC-MS analyses of elution and 'reference' samples, and quantitation were performed as in Affinity capture of 12ca5-binding peptides-effect of ligand concentration. (Note: raw ion counts were not normalized, as PRTC was absent from the 'reference' samples. Counts for a PRTC ion are shown in Supplementary Fig. 10 to illustrate the degree of run-to-run variability in MS response.) 'Indirect capture': 1 mL solutions containing 1 mg/mL BSA, 0.02% Tween 20, 1x PBS, and 1 nM/peptide 12ca5 control binders (Supplementary Table 1) were prepared in 1.7 mL plastic centrifuge tubes. The solutions were chilled on ice for 10 min, 12ca5-biotin was added to either 100 (100 pmol) or 1 µM (1 nmol), and the resulting solutions were kept on a rotating vertical mixer (in 4 °C cold room). After 1 h, 1 mg (0.13 nmol IgG binding capacity) or 10 mg (1.3 nmol IgG binding capacity) portions of MyOne Streptavidin T1 Dynabeads were added in 100 µL each of 1 mg/mL BSA, 0.02% Tween 20, 1x PBS. The resulting solutions were kept for 15 min (rotating vertical mixer, in 4 °C cold room).
Elution, nanoLC-MS analyses of elution and 'reference' samples, and quantitation were performed as in Affinity capture of 12ca5-binding peptides-effect of ligand concentration. (Note the caveat above, under 'direct capture'.)
Following removal of the N-terminal Fmoc group, the resin was washed with DMF (150 mL), and a small portion was transferred to a plastic fritted syringe, washed with DCM (~ 10 mL), and dried under reduced pressure. 1.0 mg of resin was weighed into a plastic tube (theory: 4.7 x 10 4 beads) and set aside for later characterization (described in Characterization of 2 x 10 6 , 2 x 10 7 , and 2 x 10 8 -member (X)9K-CONH2 libraries). The remainder was resuspended in DMF and pooled back with the bulk of the library.

Portioning:
Resin was suspended in DMF (~50 mL) and divided evenly among 11 x 10 mL fritted plastic syringes. One of these portions of resin was held aside, and the remainder pooled back together (theory: 1.8 x 10 8 beads). The portion that was held aside was further divided evenly among 11 x 10 mL fritted plastic syringes. One of these portions was in turn held aside (theory: 1.7 x 10 6 beads), and the remainder was pooled back together (theory: 1.7 x 10 7 beads). These three portions of resin represent approximate 2 x 10 8 , 2 x 10 7 , and 2 x 10 6 -member libraries. Resins were each washed with DCM (~50 mL) in fritted plastic syringes and dried under reduced pressure.

Cleavage from resin:
Libraries were globally deprotected and cleaved from resin by treatment of dry resin with a solution of 94% (v/v) TFA, 2.5% (v/v) ethanedithiol, 2.5% (v/v) water, and 1.0% (v/v) triisopropylsilane, for 3 h at ambient temperature. TFA was then evaporated under a stream of nitrogen, and crude peptide was precipitated by addition of cold diethyl ether. Precipitated peptide was triturated (3x) with cold diethyl ether, dissolved in 30/70 water/acetonitrile (0.1% TFA), passed through a 0.2 μm PTFE syringe filter, and lyophilized.

Preparation of stock solutions:
Lyophilized powder of 2 x 10 8 -member library (106 mg) was dissolved in DMF (1.08 mL) and then diluted with 1x PBS (9.76 mL) to bring the final concentration to ~8 mM total peptide (~40 pM/member). Lyophilized powder of 2 x 10 7 -member library (39 mg) was dissolved in DMF (3.96 mL) and diluted with 1x PBS (35.6 mL) to bring the final concentration to ~0.8 mM total peptide (~40 pM/member). Lyophilized powder of 2 x 10 6 -member library was dissolved in DMF (1.09 mL) and diluted with 1x PBS (9.83 mL) to bring the final concentration to ~0.8 mM total peptide (~400 pM/member). Stock solutions were aliquotted out and stored at -80 °C. Aliquots were thawed on ice prior to use.

NanoLC-MS/MS analysis:
Details of the columns and instruments used for analysis are provided in Affinity capture of 12ca5-binding peptides-effect of ligand concentration. The standard nano-LC method was run at 40 °C and a flow rate of 300 nL/min with the following gradient: 1% solvent B in solvent A ramping linearly to 41% B in A over 120 min, where solvent A = water (0.1% FA), and solvent B = 80% acetonitrile, 20% water (0.1% FA). Positive ion spray voltage was set to 2200 V. Orbitrap detection was used for primary MS with the following parameters: resolution = 120,000; quadrupole isolation; scan range = 200-1400 m/z; RF lens = 30%; AGC target = 1 x 10 6 ; maximum injection time = 100 ms; 1 microsan.
Acquisition of secondary MS spectra was done in a data-dependent manner: dynamic exclusion was employed such that a precursor was excluded for 30 s if it was detected four or more times within 30 s (mass tolerance: 10.00 ppm); monoisotopic precursor selection used to select for peptides; intensity threshold was set to 5 x 10 4 ; charge states 2-10 were selected; and precursor selection range was set to 200-1400 m/z. The top 15 most intense precursors that met the preceding criteria were subjected to subsequent fragmentation.
Three fragmentation modes -collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron-transfer/higher-energy collisional dissociation (EThcD) -were used for acquisition of secondary MS spectra. Only precursors with charge states 3 and above were subjected to all three fragmentation modes; precursors with charge states of 2 were subjected to CID and HCD only. For all three modes, detection was performed in the Orbitrap (resolution = 30,000; quadrupole isolation; isolation window = 1.3 m/z; AGC target = 2 x 10 4 ; maximum injection time = 100 ms; 1 microscan). For CID, a collision energy of 30% was used. For HCD, a collision energy of 25% was used. For EthcD, a supplemental activation collision energy of 25% was used.

De novo peptide sequencing:
De novo peptide sequencing was performed by processing .raw files obtained from Orbitrap analysis using PEAKS Studio (version 8.5) from Bioinformatics Solutions Inc. (ON, Canada). HCD and CID scans were merged within a 0.2 minute and 0.02 Da window, mass precursor correction was used, and primary mass filtration was employed as appropriate. Auto de novo sequencing was performed using a 15 ppm precursor mass error and 0.02 Da fragment mass error, and with the following modifications: fixed Cterm amidation (-0.98 Da) on lysine, and variable oxidation on methionine (+15.99 Da). 15 candidate sequences were obtained for each preprocessed scan. Post-de novo data analysis was performed as described in Vinogradov Affinity selections of 2 x 10 6 , 2 x 10 7 , and 2 x 10 8 -member libraries against 12ca5: Preparation of 12ca5-functionalized magnetic beads: MyOne Streptavidin T1 Dynabeads (300 μL of 10 mg/mL stock) were transferred to 1.7 mL plastic centrifuge tubes, and placed in a magnetic separation rack. Beads were washed 3 x 1 mL w/ 10% FBS, 0.02% Tween 20, 1x PBS, and then treated with 300 μL of biotinylated 12ca5 (1.5 μM; 0.45 nmol). The resulting suspensions were transferred to a rotating vertical mixer and allowed to incubate for 1 h at 4°C. After this time, the beads were returned to the separating rack, the supernatant was removed, and the beads were washed 3 x 1 mL w/ 10% FBS, 0.02% Tween 20, 1x PBS. Beads were resuspended in 300 μL of 10% FBS, 0.02% Tween 20, 1x PBS.

Elution:
The centrifuge tubes containing the bead suspensions were transferred to the magnetic separation rack. The beads were washed 3 x 1 mL w/ 1x PBS. Bound peptides were eluted with 2 x 100 μL 6M guanidine hydrochloride, 200 mM phosphate, pH 6.8. Eluates were concentrated via C18 ZipTip® pipette tips (as described in Affinity capture of 12ca5-binding peptides-effect of ligand concentration, with concentration of eluate) and lyophilized.

SPPS of HA epitope Ala scan mutants:
Peptides were synthesized on a fully automated fast-flow peptide synthesizer as described in Solid phase peptide synthesis (SPPS) of anti-hemagglutinin (HA) epitope and analogues. Concomitant side chain deprotection and cleavage from resin, as well as HPLC purification and LC-MS analysis, were also carried out as described in Solid phase peptide synthesis (SPPS) of anti-hemagglutinin (HA) epitope and analogues.

Competition fluorescence polarization of HA epitope Ala scan mutants:
Competition fluorescence polarization experiments were carried out as described in Competition fluorescence polarization of HA epitope and analogues.
Following removal of the N-terminal Fmoc group, the resin was washed with DMF (150 mL), and a small portion was transferred to a plastic fritted syringe, washed with DCM (~ 10 mL), and dried under reduced pressure. 1.0 mg of resin was weighed into a plastic tube (theory: 1.6 x 10 5 beads) and set aside for later characterization (described in Characterization of a 1 x 10 9 -member (X)9K-CONH2 library). The remainder was resuspended in DMF and pooled back with the bulk of the library.

Portioning:
Resin was suspended in DMF (~50 mL) and divided evenly among 11 x 10 mL fritted plastic syringes. One of these portions of resin was held aside (theory: 1.2 x 10 8 beads), and the remainder pooled back together (theory: 1.2 x 10 9 beads). These two portions of resin represent approximate 1 x 10 9 and 1 x 10 8 -member libraries. Resins were each washed with DCM (~50 mL) in fritted plastic syringes and dried under reduced pressure.
NanoLC-MS/MS analysis and de novo peptide sequencing: Analysis and de novo sequencing was performed as described in Characterization of 2 x 10 6 , 2 x 10 7 , and 2 x 10 8 -member (X)9K-CONH2 libraries.
Affinity capture: Preparation of 12ca5-functionalized magnetic beads: MyOne Streptavidin T1 Dynabeads (1.1 mL of 10 mg/mL stock) were transferred to a 1.7 mL plastic centrifuge tube, and placed in a magnetic separation rack. Beads were washed 3 x 1 mL w/ 10% FBS, 1x PBS, and then treated with 1.0 mL of biotinylated 12ca5 (1.7 μM; 1.7 nmol). The resulting suspensions were transferred to a rotating vertical mixer and allowed to incubate for 1 h at 4°C. After this time, the beads were returned to the separating rack, the supernatant was removed, and the beads were washed 3 x 1 mL w/ 10% FBS, 1x PBS. Beads were resuspended in 300 μL of 10% FBS, 1x PBS.

Portioning:
Following removal of the N-terminal Fmoc group, the resin was washed with DMF (150 mL), then suspended in DMF (~ 50 mL) and divided evenly among 5 x 20 mL fritted plastic syringes. Four of these portions (theory: 2 x 10 8 beads each) were washed with DCM (3x) and dried under reduced pressure. The fifth portion was further divided among 11 x 10 mL fritted plastic syringes. Ten of these portions were recombined. The recombined beads, along with the remaining portion (theory: 1.8 x 10 7 beads), were washed with DCM (3x) and dried under reduced pressure. 1.0 mg of dried resin was weighed into a plastic tube (theory: 1.4 x 10 5 beads) and set aside for later characterization (described in Characterization of a 1 x 10 9 -member (X)12K-CONH2 library).

Preparation of stock solutions:
Lyophilized powders of 2 x 10 8 -member libraries were each dissolved first in DMF and then diluted with 1x PBS to a final library concentration of 8 mM (~40 pM/member), and a final DMF concentration of 10% (v/v). Lyophilized powder of 2 x 10 7 -member library was similarly first dissolved in DMF, and then diluted with 1x PBS to a final library concentration of 7 mM (~400 pM/member), and a final DMF concentration of 10% (v/v). Stock solutions were aliquotted out and stored at -80 °C. Aliquots were thawed on ice prior to use.
NanoLC-MS/MS analysis and de novo peptide sequencing: Analysis and de novo sequencing was performed as described in Characterization of 2 x 10 6 , 2 x 10 7 , and 2 x 10 8 -member (X)9K-CONH2 libraries.
A biotin was site-specifically incorporated as follows: Fmoc-L-Lys(alloc)-OH was used for coupling of Lys36 during SPPS. Following main chain elaboration, the N-terminal amino group was Boc-protected by first washing the resin 3 times with DCM, then adding to the resin a solution of di-tert-butyl dicarbonate (40 eq, 400 mM) and DIEA (40 eq) in DCM. Coupling was allowed to proceed for 30 min. At this time, resin was washed 5 times with DCM and coupling was repeated as described.

Expression of 14-3-3γ:
Full-length human 14-3-3γ was expressed by transforming pROEX HTb plasmid, containing a His6-tagged 14-3-3γFL gene and ampicillin resistance gene, to Rosetta(DE3) Escherichia coli cells (Novagen). Cells were grown at 37 °C, with 0.1 mg/mL ampicillin, and protein expression was induced using 0.4 μM IPTG and 1 mM MgCl2 and left overnight at 18 °C. Cells were harvested and resuspended in 200 mL of wash buffer (50 mM Tris, 300 mM NaCl, 12.5 mM imidazole, 2 mM β-mercaptoethanol (BME), pH = 8.0). The proteins were isolated by homogenizing the cell pellets at a pressure of 40 psi using Emulsiflex-C3 homogenizer. The homogenized mixture was centrifuged at 40,000 x g for 30 min at 4 °C and the supernatant was loaded onto a nickel-nitrilotriacetic acid affinity column (Qiagen) pre-equilibrated with wash buffer. After washing the column with wash buffer containing 12.5 mM imidazole, the bound protein was eluted with 250 mM imidazole. Fractions containing protein were verified using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The protein containing fractions was dialyzed into dialysis buffer (50 mM Tris pH 8, 300 mM NaCl and 2 mM BME) and in a next step to ITC buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2 and 0.5 mM TCEP). The protein was concentrated (measured using Nanodrop-1000), aliquotted and stored at -80 °C.
Purity and exact mass of the 14-3-3γ protein was determined using a High Resolution LC-MS system consisting of a Waters ACQUITY UPLC I-Class system coupled to a Xevo G2 Quadrupole Time of Flight (Q-ToF). The system was comprised of a Binary Solvent Manager and a Sample Manager with Fixed-Loop (SM-FL). The protein was separated (0.3 mL/min) by the column (Polaris C18A reverse phase column 2.0 x 100 mm, Agilent) using a 15% to 75% acetonitrile gradient in water (0.1% v/v formic acid) before analysis in positive mode in the mass spectrometer. Deconvolution was performed using the MaxENTI algorithm in the Masslynx v4.1 (SCN862) software.
At this stage, resin was suspended in DMF (50 mL), and divided evenly among 18 x 10 mL fritted plastic syringes using a 5 mL Eppendorf pipette. Couplings were performed as follows: Fmoc-protected amino acids (0.29 mmol) in HATU solution , activated with DIEA (1.9 mL, 11 mmol) immediately prior to coupling, and added to resin bed. Coupling was performed for 2.5 h; after this time, resin was washed with DMF (100 mL). Fmoc removal was carried out by treatment of resin with 20% piperidine in DMF (1 x 50 mL flow wash; 2 x 50 mL, 5 min batch treatments). Resin was then washed with DMF (150 mL). Four more cycles of split-and-pool synthesis were then performed as described above.
Following removal of N-terminal Fmoc group, resin was washed with DMF (150 mL) and transferred to fritted plastic syringes (20 mL). Resin was then washed with DCM (3x) and dried under reduced pressure. 1.0 mg of dried resin (theory: 4.8 x 10 4 beads) was weighed into a plastic tube and set aside for later characterization (Characterization of a non-canonical (X)4(pS)(X)4-CONH2 library).

Preparation of stock solution:
Lyophilized powder of library (123 mg) was dissolved in DMF (1.16 mL) and diluted with 1x PBS (10.5 mL) to a final library concentration of 8 mM (~40 pM/member) and final DMF concentration of 10% (v/v). Stock solutions were aliquotted out and stored at -80 °C. Aliquots were thawed on ice prior to use.

NanoLC-MS/MS analysis and de novo peptide sequencing:
Analysis and de novo sequencing was performed as described in Characterization of 2 x 10 6 , 2 x 10 7 , and 2 x 10 8 -member (X)9K-CONH2 libraries. Non-canonical amino acids with masses that differ from natural amino acids were sequenced as fixed modifications on residues that had been excluded from the monomer set. Specifically, β-homothreonine was identified as fixed modification on Asn (+1.0204), aminoadipic acid as a fixed modification on Glu (+14.0156), diaminobutyric acid as a fixed modification on Gly (+43.0421), ornithine as a fixed modification on Cys (+11.0701), hydroxyproline as a fixed modification on Pro (+15.9948), cyclopropyl alanine as a fixed modification on Met (-19.9721), cyclohexyl alanine as a fixed modification on Phe (+6.0469), 4-amino phenylalanine as a fixed modification on Arg (+5.9782), 4-fluoro phenylalanine as a fixed modification on Tyr (+1.9957), 4-nitro phenylalanine as a fixed modification on Trp (+5.9742), thiazolyl alanine as a fixed modification on His (+16.9611), and phosphoserine as a fixed modification on Ser (+79.9663). β-alanine, β-homoserine, and norvaline were identified as Ala, Thr, and Val, respectively. D-Leu, D-Lys, D-Asp, and D-Gln were identified as Leu, Lys, Asp, and Gln, respectively.

Preparation of Lys(boc)-β-Ala-Lys(alloc)-Rink amide peptidyl resin:
Rink amide ChemMatrix resin (1.0 g, 0.45 mmol/g) was transferred to a 20 mL plastic fritted syringe, washed 3 x 20 mL with DMF, and swollen in 20 mL of DMF for 1 h. Fmoc-L-Lys(alloc)-OH (2.25 mmol, 5 eq, 1.0 g) was weighed into a glass vial and dissolved in 0.38 M HATU in DMF (5.34 mL, 2.0 mmol, 0.9 eq HATU). To this solution was added DIEA (1.13 mL, 6.5 mmol, 2.9 eq), and activated amino acid solution was added to the resin bed. Coupling was allowed to proceed for 1 h. At this time, the reaction mixture was drained and the resin was washed 3 x 20 mL with DMF. Fmoc removal was carried out by treatment of the resin with 20% piperidine in DMF (2 x 20 mL, 5 min batch treatments). Resin was then washed 3 x 20 mL with DMF. Couplings of Fmoc-β-Ala-OH and Fmoc-Lys(boc)-OH were performed in the same manner. After removal of the N-terminal Fmoc group, the resin was suspended in DMF (~20 mL) and split out 10 ways into 6 mL plastic fritted syringes. (Note: β-Ala was incorporated as a spacer between the sequences obtained from selection, which all bear a C-terminal lysine, and Lys(alloc), to which a FITC fluorophore will be coupled for fluorescence anisotropy studies.) Main chain elaboration of select sequences derived from affinity selection: One portion of peptidyl resin prepared above was used for every construct prepared (four in total). Couplings were performed as follows: Fmoc-protected amino acids (0.23 mmol) in HATU solution (0.38M, 534 μL, 0.2 mmol) were activated with DIEA (113 μL, 0.65 mmol) and added to the resin bed. Couplings were allowed to proceed for 20 min. At this time, reaction mixtures were drained and resins were washed 3 x 5 mL with DMF. Fmoc removal was carried out by treatment of the resin with 20% piperidine in DMF (1 x 5 mL flow wash; 2 x 5 mL, 5 min batch treatments). Following removal of N-terminal Fmoc group, resins were washed 3 x 5 mL with DMF, then 3 x 5 mL with DCM.

Incorporation of FITC:
The free amine on the N-terminus was Boc-protected as follows: to a solution of di-tert-butyl dicarbonate (0.45 mmol, 10 eq, 400 mM) in DCM was added DIEA (10 eq), and solution was added to each portion of resin. Coupling was allowed to proceed for 1 h. At this time, resin was washed 3 x 5 mL with DCM and coupling was repeated as described. Resin was washed 5 x 5 mL with DCM.
Alloc removal was achieved as follows: each portion of resin was treated with a solution of tetrakis(triphenylphosphine)palladium(0) (0.5 eq, 20 mM) and phenylsilane (20 eq) in DCM, 2 x 45 min. Resins were then washed 3 x 5 mL with DCM, then 3 x 5 mL with DMF.
FITC was installed on the free amine on each C-terminal lysine by treating each portion of resin with fluorescein isothiocyanate isomer I (10 eq, 400 mM in 4:1 DMF:DCM) and DIEA (15 eq) for 1.5 h. Reactions were kept under aluminum foil for the duration of the coupling. Reaction mixtures were then drained, and resins were washed 3 x 5 mL with DMF, 3 x 5 mL with DCM, and dried under reduced pressure.

Cleavage from resin, HPLC purification, and LC-MS characterization:
Detailed procedures can be found in Solid phase peptide synthesis (SPPS) of anti-hemagglutinin (HA) epitope and analogues. Sequences, structures, and LC-MS traces are shown below.

SPPS of unlabeled 14-3-3γ-binding peptides:
SPPS was carried out on Rink amide ChemMatrix resin (0.45 mmol/g). Couplings for main chain elaboration were carried out as described in SPPS of FITC-labeled putative 14-3-3γ-binding peptides. Cleavage from resin, HPLC purification, and LC-MS characterization were performed as described in Solid phase peptide synthesis (SPPS) of anti-hemagglutinin (HA) epitope and analogues. Sequences, structures, and LC-MS traces are shown below.

Expression of 14-3-3σΔc:
The 14-3-3σ isoform with a truncated C-terminus after T321 (ΔC, to enhance crystallization) was expressed by transforming pROEX HTb plasmid, containing a His6tagged 14-3-3σΔc gene and ampicillin resistance gene, to BL21(DE3) Escherichia coli cells (Novagen). Cells were grown at 37 °C, with 0.1 mg/mL ampicillin, and protein expression was induced using 0.4 μM IPTG and 1 mM MgCl2 and left overnight at 18 °C. Cells were harvested and resuspended in 200 mL of wash buffer (50 mM Tris, 300 mM NaCl, 12.5 mM imidazole, 2 mM β-mercaptoethanol (BME), pH = 8.0). The proteins were isolated by homogenizing the cell pellets at a pressure of 40 psi using Emulsiflex-C3 homogenizer. The homogenized mixture was centrifuged at 40,000 x g for 30 min at 4 °C and the supernatant was loaded onto a nickel-nitrilotriacetic acid affinity column (Qiagen) pre-equilibrated with wash buffer. After washing the column with wash buffer containing 12.5 mM imidazole, the bound protein was eluted with 250 mM imidazole. Fractions containing protein were verified using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The protein containing fractions were dialyzed overnight in dialysis buffer (50 mM Tris pH 8, 300 mM NaCl and 2 mM BME) containing TEV protease for His-tag cleavage at 4°C. Non-cleaved His-tagged 14-3-3σΔc was than captured using a nickel-nitrilotriacetic acid affinity column (Qiagen) preequilibrated with wash buffer after which the flow through was dialyzed into ITC buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2 and 0.5 mM TCEP) at 4°C. The protein was concentrated (measured using Nanodrop-1000), aliquotted and stored at -80 °C. Purity and exact mass of the 14-3-3σΔc protein was determined as described in Expression of 14-3-3γ.
Integration, scaling and merging of data was done using DIALS (CCP4i2) after which molecular replacement is done with MOLREP (CCP4i2) using PDB 4JC3 as search model. A three-dimensional structure of 14-3-3.12 was generated using eLBOW (Phenix) 6 after which it was built within this structure based on visual inspection of Fo-Fc and 2FoFc electron density maps in Coot 7 . Several rounds of model building and refinement (based on isotropic b-factors and standard set of stereo-chemical restraints: covalent bonds, angles, dihedrals, planarities, chiralities, non-bonded) were performed using Coot and Phenix.refine 8,9 . See Supplementary Table 12 for data collection and refinement statistics.  Table 1), only the 2 highest-affinity binders were significantly retained. Error bars correspond to the standard deviations in recovery obtained by 3 technical replicates.
Supplementary Figure 9. High-affinity 12ca5-binding peptides are efficiently recovered with 'direct' capture at high dilution. Pulldowns were performed using 100 nM 12ca5 and the indicated ligand concentrations. Recoveries were determined by nLC-MS analysis of the peptide mixtures obtained by 'direct' or 'indirect' capture, relative to a reference analysis (the amount of material corresponding to 100% retention). Raw ion counts were normalized to an internal standard to account for run-to-run variability in MS response.  Extracted ion chromatograms (EICs) for peptides that were sequenced in 12ca5 selections, but not IgG1 selections, and which may be 12ca5 binders but were possibly mis-sequenced. All peptides shown contain either DXXDFS or DXXDSF. Because the dipeptide masses of 'FS' and 'YA' are identical, incomplete fragmentation could have led to erroneous sequence assignments in these cases. The EICs indicate that these peptides were enriched in 12ca5 selections, suggesting that they could in fact be 12ca5-binding peptides. Supplementary Figure 26. EICs for peptides that were sequenced in 12ca5 selections, but not IgG1 selections, and which do not contain the HA epitope. The presence of these peptides is detected in both 12ca5 and IgG1 selections, indicating that these binders are non-specific, despite only having been sequenced from 12ca5 selections. Extracted ion chromatograms (EICs) of a subset of 14-3-3γ-unique sequences reveal these peptides were retained in each selection replicate against 14-3-3γ (blue traces). Signals for these peptides are also absent in the chromatograms from 12ca5 selections (orange traces), suggesting they were pulled down to the presence of 14-3-3. Three peptides are examined here: a) 14.3.3.1, b) 14-3-3.6, and c) 14-3-3.12 (Supplementary Table 11).    Supplementary Figure 42. FITC-labeled 14-3-3.12 retains binding activity for 14-3-3σ as measured by fluorescence anistropy, with 5 to 12-fold reduced affinity relative to that for 14-3-3γ. Measurements were taken either immediately after, 4 h after, or 20 h after incubation of 14-3-2.12 with 14-3-3. Error bars correspond to standard error among three technical replicates.  List of all sequences (ALC score ≥ 80) identified from selections performed with 2 x 10 6 , 2 x 10 7 , and 2 x 10 8 -member libraries against 12ca5 (one replicate shown). Sequences are separated by the library they were discovered from and listed by decreasing ALC score.

Supplementary
Supplementary Table 3. Replicate selections from a 2 x 10 8 -member library identify similar populations of 12ca5-binding peptides.
Out of three replicate selections from a 2 x 10 8 -member library for 12ca5 binding, approximately 60% of DXXDY(A/S)-containing sequences are identified in multiple replicates, suggesting that similar (although not identical) populations of 12ca5-binding peptides are reproducibly identified. In total, 150 DXXDY(A/S)-containing sequences were obtained. At low sample loading, use of a precursor selection threshold of 5 x 10 4 yielded only one identified sequence (MNDLVDYADK). At high sample loading, two additional DXXDY-containing sequences were identified, along with 32 non-motif-containing sequences. MS signal is reported as the apex of extracted ion chromatograms.  Supplementary Table 5. Selections against 12ca5 identify a decreasing number of motif-containing sequences as library size is increased from 10 8 to 10 9 .
An approximate 9-fold drop in the number of 12ca5-binding sequences is observed from one-pot selections of a 10 9 -member library relative to a 10 8 -member library. Selections were performed near the solubility limit of the libraries (1-2 mM), and variable member concentration. Supplementary Table 6. Increasing scale of 10 9 -member library selections does not restore recovery of 12ca5-binding peptides.
An increase in the amount of each library member present in the selection did not yield an increase in the number of sequences bearing the characteristic DXXDY(A/S) motif, suggesting that the decreased number of 12ca5-binding sequences identified is not due to material limitation.  Table 7. Ten-fold increase in 12ca5 in selections from a 10 9 -member library does not restore recovery of 12ca5-binding peptides.
Shown are the number of DXXDY(A/S)-containing sequences and total sequences identified from side-by-side selections of a 2 x 10 8 -member library against 12ca5 and polyclonal human IgG1. Selections were performed in triplicate, and unique sequences from the sum of these technical replicates are indicated. Subtracting out non-specific sequences (those identified in both conditions) improves identification of motifcontaining sequences as a fraction of the total.