PEPperMAP ® Substitution Scan of Epitope GVPEQEDSVLFR against Mouse Monoclonal Antibody 7B10 PEPperPRINT GmbH Heidelberg, 10/2013

Experimental Conditions Microarray Content:Full substitution scan of epitope GVPEQEDSVLFR with 20 standard amino acids, citrulline (coded as “Z”) and two D-amino acids (D-alanine and D-glutamic acid; coded as dA/a and dE/e); the corresponding peptide microarray contained 265 different peptides printed at least as duplicates resulting in 552 peptides spots; the peptide microarray was further framed by additional HA and Flag control peptides (60 and 62 spots, respectively). Sample: Mouse monoclonal antibody 7B10 (anti-human SMN antibody 1 ; immunoGlobe Antikörpertechnik GmbH, Himmelstadt, Germany); staining in incubation buffer at a concentration of 0.2 µg/ml for 16 h at 4°C and slight shaking at 500 rpm. Washing Buffer:PBS, pH 7.4 with 0.05% Tween 20 (2x1 min after each assay) Blocking Buffer:Rockland blocking buffer MB-070 (60 min before the first assay) Incubation Buffer: PBS, pH 7.4 with 0.05% Tween 20 and 10% Rockland blocking buffer Secondary Antibody: Goat anti-mouse IgG (H+L) DyLight680 antibody; 30 min staining in incubation buffer at RT and a dilution of 1:5000 Control Antibodies: Monoclonal anti-HA (12CA5)-LL-DyLight680 (1:1000), monoclonal anti-FLAG(M2)-LL- DyLight800; staining in incubation buffer for 1 h at RT and a dilution of 1:1000 Scanner: LI-COR Odyssey Imaging System; scanning offset 1 mm, resolution 21 µm, scanning intensity of 5 (main assay, red) and 7/7 (control assay, green/red) Microarray Data Files:MicroarrayData_Mouse_mAb_7B10.xlsx Microarray Identifier:000586_04 (10 nm copolymer coated glass slides with β-alanine-aspartic acid linker) 1 M. Kroiss et al., Fly (Austin) 2009; 3(3):221-8.

Data Analysis Pre-staining of one of peptide array was done with the goat anti-mouse IgG(H+L) conj. DyLight680 antibody at a dilution of 1:5000 to investigate background interactions of the GVPEQEDSVLFR variants that could interfere with the main assays. Subsequent incubation of the peptide microarray with mouse monoclonal antibody 7B10 at a concentration of 0.2 µg/ml in incubation buffer was followed by staining with the secondary antibody and read-out at a scanning intensity of 5 (red). Finally, HA and Flag control peptides framing the peptide arrays were stained with high and homogeneous spot intensities (scanning intensities green/red: 7/7). Quantification of spot intensities and peptide annotation were done with PepSlide ® Analyzer and summarized in the microarray data file MicroarrayData_Mouse_mAb_7B10.xlsx. A software algorithm breaks down fluorescence intensities of each spot into raw, foreground and background signal (see “Mapping Raw Data” tab), and calculates the standard deviation of foreground median intensities (see “Mapping Summary” tab). Based on averaged foreground median intensities, intensity maps were generated and binders in the peptide map highlighted by an intensity color code with red for high and white for low spot intensities. To provide an in-depth view on the permutation scan, we further generated heatmaps of the microarray as well as substitution matrices and amino acid plots reflecting the amino acid preferences at a given position. The data sets were correlated with peptide and intensity maps as well as visual inspection of the microarray scans to analyze the permutation pattern in consideration of sequence conservation and possible amino acid exchanges. The substitution matrices highlighted the preference for a given amino acid by color codes (red: preferred amino acids; green: less preferred amino acids) and were calculated by dividing the spot intensity of a given peptide (e.g. 1 YPYDVQDYA 9 ) by the averaged spot intensities of all 20 peptides that were substituted at the same position ( 1 YPYDVXDYA 9 ).The amino acid plots were calculated by dividing the spot intensity of a given peptide (e.g. 1 YPYDVQDYA 9 ) by the spot intensity of the native epitope ( 1 YPYDVPDYA 9 ). The position of an amino acid at a given position thus reflected the intensity ratio compared to the amino acid of the native epitope at the same position.

Microarray Scans After 10 min pre-swelling in standard buffer and 60 min in blocking buffer, the peptide microarray was initially incubated with the secondary goat anti-mouse IgG (H+L) DyLight680 antibody at a dilution of 1:5000 for 60 min at room temperature. At a scanning intensity of 7, we did not observed any background interaction with the GVPEQEDSVLFR variants on the microarrays due to non-specific binding of the secondary antibody (left). Subsequent Incubation of the peptide microarray with mouse monoclonal antibody 7B10 at a concentration of 0.2 µg/ml in incubation buffer was followed by staining with the secondary antibody and read-out at a scanning intensity of 5 (red). We observed a clear and well-defined substitution pattern at high spot intensities and excellent signal to noise ratios (middle). HA and Flag control peptides framing the peptide array were finally stained by the corresponding control antibodies at high and homogeneous spot intensities (right; scanning intensity red/green: 7/7). The microarray layout was shown in the peptide map in Excel file MicroarrayData_Mouse_mAb_7B10.xlsx. The full substitution scan of epitope GVPEQEDSVLFR with 23 different amino acids was printed in single spots with two array copies next two each other. HA and Flag Control Staining Goat anti-mouse antibody, 1:5000 Mouse monoclonal antibody 7B10, 0.2 µg/ml

Data Evaluation The heatmap, the substitution matrix and the amino acid plot of the substitution scan of epitope 1 GVPEQEDSVLFR 12 highlighted a conserved core motif 3 PEQEDSVLF 11 framed by variable amino acids 1 G and 2 V at the N-terminus and 12 R at the C-terminus. Most conserved amino acid was 11 F that did not tolerate substitution with any other amino acid without complete loss in binding. Amino acids 10 L and 8 S were also highly conserved with a tolerance for conservative exchange with I ( 10 L) and T ( 8 S) as well as to a lesser extent with V ( 10 L) and G ( 8 S). Amino acids 9 V and 7 D were also well conserved, but could be exchanged conservatively with I ( 9 V) and E ( 7 D) as well as with T ( 9 V) and C ( 7 D) with 50% - 75% reduction of spot intensities. Amino acids 6 E and 4 E were poorly conserved, but exhibited a slight objection for replacement with amino acids e, H, P, W and Z. In contrast to this amino acids 5 Q and 3 P were well conserved with a certain tolerance for exchange with hydrophobic amino acids V and I ( 3 P) as well as with P ( 5 Q) and also some other amino acids like A, M and K to a lesser extent. Except for a position 6 E, replacement of amino acids in the conserved core motif 3 PEQEDSVLF 11 with D-amino acids a and e was hardly tolerated.

Discussion & Conclusion The substitution scan of epitope 1 GVPEQEDSVLFR 12 with 20 standard amino acids, citrulline (coded as “Z”) and two D-amino acids (D-alanine and D-glutamic acid; coded as dA/a and dE/e) was done against mouse monoclonal antibody 7B10 (anti- human SMN antibody; antigen human survival of motor neuron protein) at a concentration of 0.2 µg/ml in incubation buffer. Staining with the secondary goat anti-mouse IgG (H+L) DyLight680 antibody was followed by read-out with a LI-COR Odyssey Imaging System at scanning intensities of 5 (main assay) and 7/7 (control staining). Quantification of spot intensities and peptide annotation were done with PepSlide ® Analyzer. Incubation of the peptide microarray with mouse monoclonal antibody 7B10 resulted in high spot intensities and excellent signal to noise ratios at a rather low concentration of 0.2 µg/ml in incubation buffer. The substitution scan of 1 GVPEQEDSVLFR 12 clearly revealed a conserved core motif 3 PEQEDSVLF 11 with highly conserved amino acids 11 F, 10 L and 8 S as well as well-conserved positions 9 V, 7 D, 5 Q and 3 P. In contrast to this, amino acids 6 E and 4 E were poorly conserved. Exchange of both positions with S caused an increase in spot intensities and hence binding of mouse monoclonal antibody 7B10. Except for exchange of 6 E with a, D-amino acids a and e were hardly tolerated in the conserved core motif, while replacement of variable positions 1 G, 2 V and 12 R was accepted. Citrulline was also not found among the preferred amino acids at any position of starting peptide 1 GVPEQEDSVLFR 12, but less refused compared to both D-amino acids. PEPperMAP ® substitution scans of given epitopes are available as full service offer for less than 1,500 Euro per sample. On request PEPperPRINT can also run permutation scans with more D-amino acids to identify possible non-natural epitope variants. PEPperMAP ® substitution scans of given epitopes are also available with the simultaneous substitution of two amino acid positions along full or partial peptide sequences.

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