Production of high-complexity frameshift neoantigen peptide microarrays

Abstract

Parallel measurement of large numbers of antigen-antibody interactions are increasingly enabled by peptide microarray technologies. Our group has developed an in situ synthesized peptide microarray of >400 000 frameshift neoantigens using mask-based photolithographic peptide synthesis, to profile patient specific neoantigen reactive antibodies in a single assay. The system produces 208 replicate mircoarrays per wafer and is capable of producing multiple wafers per synthetic lot to routinely synthesize over 300 million peptides simultaneously. In this report, we demonstrate the feasibility of the system for detecting peripheral-blood antibody binding to frameshift neoantigens across multiple synthetic lots.

Fig. 1. Schematic of photolithographic peptide synthesis process (top) and peptide microarrays produced per silicon wafer (bottom).

Fig. 2. Analysis of normal donor serum on 409 600 peptide microarray. (a) Cell plot of each peptide array (column) by each peptide (row) where red indicates high binding and blue indicates low binding. (b) Correlation between replicate peptide arrays versus serum dilution. Red indicates high correlation and blue indicates low correlation. (c) The maximum observed signal (red squares), 99.5 percentile (blue circles) and the peptide array median (black circles) for the averaged RFU as a function of serum dilution.

Fig. 3. Identification of dose responsive peptides from a serum sample. (a) Scatterplot of mean RFU of triplicate arrays for 1 : 100 (y-axis) versus 1 : 800 (x-axis) dilution with each data point colored by mean RFU for the 1 : 2400 dilution (color bar) where red indicates high binding and blue indicates low binding. (b) Average RFU from triplicate arrays as a function of serum dilution for 225 dose responsive peptides.

Fig. 4. Mixing effects in IgG–peptide binding. (a) Distribution of RFU values for 4752 replicate spots of APLARPRSPAPAA (left) and 4702 spots of APLRRGRSWIMPSSF (right) for 1 : 100 dilution (red), 1 : 800 dilution (blue), and 1 : 2400 dilution (black). Difference in RFU from the array mean for each replicate peptide (y-axis) versus column position within the microarray (x-axis) at (b) the 1 : 800 dilution for APLARPRSRPAPAA and (c) the 1 : 100 dilution for APLRRGRSWIMPSSF. Each spot is coloured according to its RFU.

Fig. 5. Plot of peptide mean RFU (y-axis) across 18 arrays versus the standard deviation (x-axis) for 4 096 000 peptides. The positive rate of each peptide is indicated on the color scale bar from 100% positive (red) down to 0% positive (blue).