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 antibody1; 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. 1YPYDVQDYA9) by the averaged

spot intensities of all 20 peptides that were substituted at the same position (1YPYDVXDYA9).The amino acid plots were calculated

by dividing the spot intensity of a given peptide (e.g. 1YPYDVQDYA9) by the spot intensity of the native epitope (1YPYDVPDYA9).

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 StainingGoat 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 1GVPEQEDSVLFR12 highlighted a conserved core motif 3PEQEDSVLF11 framed by variable amino acids 1G and 2V at the N-terminus and 12R at the C-terminus. Most conserved amino acid was 11F that did not tolerate substitution with any other amino acid without complete loss in binding. Amino acids 10L and 8S were also highly conserved with a tolerance for conservative exchange with I (10L) and T (8S) as well as to a lesser extent with V (10L) and G (8S). Amino acids 9V and 7D were also well conserved, but could be exchanged conservatively with I (9V) and E (7D) as well as with T (9V) and C (7D) with 50% - 75% reduction of spot intensities.

Amino acids 6E and 4E 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 5Q and 3P were well conserved with a certain tolerance for exchange with hydrophobic amino acids V and I (3P) as well as with P (5Q) and also some other amino acids like A, M and K to a lesser extent. Except for a position 6E, replacement of amino acids in the conserved core motif 3PEQEDSVLF11 with D-amino acids a and e was hardly tolerated.

A C D E F G H I K L M N P Q R S T V W Y Z a eRFLVSDEQEPVG

A C D E F G H I K L M N P Q R S T V W Y Z a eR T A S I V P G K M F Q L E N Y a R D e C H Z WF F W K I E G S Y R D C H Z Q N T V L P A M a eL L I V T F E S M K D N W A Q R H Z P G Y C a eV V T I P L A W S E Q H Z K D R M N Y G C F a eS T S G A D Z W Q V H R E F M I e N C K Y a L PD D E C M Q S G A L N Z e W F H Y T I R K V a PE S K T A G V I M F E Y D L Q a C R Z N W e H PQ Q P A M K S W R T G D V Z N F E L Y I H C a eE V L T S I K E M D A Q F G Y R N Z C W H P a eP P V I Q L A R T H N Z G W C K S E F M Y D a eV T Q R S N V I G W P F M L Y C H E a Z D e A KG C I R E Q M K W G A S T V F H D N a L Z Y e P

Discussion & Conclusion

The substitution scan of epitope 1GVPEQEDSVLFR12 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 1GVPEQEDSVLFR12 clearly revealed a conserved core motif 3PEQEDSVLF11 with highly conserved amino acids 11F, 10L and 8S

as well as well-conserved positions 9V, 7D, 5Q and 3P. In contrast to this, amino acids 6E and 4E 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 6E with a, D-amino acids a and e were hardly tolerated in the conserved core motif, while

replacement of variable positions 1G, 2V and 12R was accepted. Citrulline was also not found among the preferred amino acids

at any position of starting peptide 1GVPEQEDSVLFR12, 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.

Contact

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info@pepperprint.comwww.pepperprint.com

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