Quantifying Proteins via Olink Proximity Assay

Proteins were measured using the Olink^® Mouse Exploratory Panel* (Olink Proteomics AB, Uppsala, Sweden) according to the manufacturer’s instructions as described previously [17 (link)]. The proximity extension assay (PEA) technology used for the Olink protocol has been well described [18 (link)] and allows 92 analytes to be analyzed simultaneously. Briefly, pairs of oligonucleotide-labeled antibody probes bind to their targeted protein, and if the 2 probes are brought in close proximity, the oligonucleotides will hybridize in a pairwise manner. The addition of DNA polymerase leads to a proximity-dependent DNA polymerization event, which generates a unique PCR target sequence. The resulting DNA sequence was subsequently detected and quantified using a microfluidic real-time PCR instrument (Biomark HD, Fluidigm, München, Germany) (Figure 6A). The data are then quality controlled and normalized using an internal extension control and an interplate control to adjust for intra- and interrun variations. The final assay read-out is presented in Normalized Protein eXpression (NPX) values, arbitrary units on a log2-scale in which a high value corresponds to high protein expression. All assay validation data (detection limits, intra- and interassay precision data, etc.) are available on the manufacturer’s website (www.olink.com; accessed date 1 May 2021).