Briefly, proteome microarrays were blocked with blocking buffer (5% BSA in 0.1% Tween 20, PBST) for 1 h at room temperature with gentle agitation. Biotin-HDCA and biotin were diluted to 100 μM in PBST and incubated on proteome microarray at room temperature for 1 h (Biotin-HDCA was synthesized by Xi'an Ruixi Biological Technology Co.,Ltd). After washing with PBST three times, microarray was incubated with 0.1% Cy5-Streptavidn solution for 20 min at room temperature in the dark, followed by three 5-min washes in PBST.
Scan protocol
The microarray was spun dry at 100 × g for 2 min and scanned with GenePixTM 4000B (Axon Instruments, CA).
Description
Biotin-HDCA was spotted on 20000 spots on human proteome microarray.
Data processing
GenePixTM Pro v6.0 was used for data analysis. The median fluorescence signal at each site (F635_Median) was divided by the background (B635_Median) for data analysis, that is, original signal strength (I) = F635_Median/B635_Median. The median and SD of I were calculated to obtain the corrected data Z-Score (corrected signal strength) for each site.
Proteins with a Z-Score greater than 2.8 were those that bind to HDCA. The mean value (I mean) represents the mean value of the original signal strength of each protein. I Mean-Ratio is the ratio between the Biotin-HDCA group and Biotin group. To call the candidates, the cutoff was set as a P value≤0.05 and I Mean-Ratio≥1.4.
