Human Proteome Microarray Assay for QPCT

The HuProt microarray assay 28 (link), 29 (link) and data analysis were performed by Wayen Biotechnologies Inc., Shanghai according to the following procedure. The HuProt microarray (CDI Laboratories, Inc.) comprises 20,240 human full-length proteins with N-terminal glutathione S-transferase (GST) tags. Human proteome microarrays (HuProtTM 20 K) were blocked with blocking buffer (1% BSA and 0.1% Tween 20 in TBST) for 1 h at room temperature with gentle agitation. The QPCT protein was labelled with biotin with an Antibody Array Assay Kit (Full Moon Biosystems, Sunnyvale, CA) and was then diluted to 0.01 mg/ml in blocking buffer and incubated on the blocked proteome microarray at room temperature for 1 h. The microarrays were washed three times for 5 min each with TBST, incubated with streptavidin-Cy5 at a dilution of 1:1000 (Thermo Fisher Scientific, USA) for 1 h at room temperature and subjected to three more 5-min washes. The microarrays were spun dry at 1500 rpm for 3 min and subjected to scanning with a GenePix 4000B (Axon Instruments, Sunnyvale, CA) to visualize and record the results. GenePix Pro 6.0 was used for data analysis. The information of all proteins contained in the HuProt microarray in the Supplementary Table 9 and the information of proteins that may bind to QPCT was provided in the Supplementary Table 10.