Obesity Intensifies Sex-specific Interferon (IFN) Signaling to Selectively Worsen Central Nervous System Autoimmunity in Females

The complete library contains antibodies targeting 1472 proteins, of which 1463 are unique proteins. Patient samples were randomized and incubated overnight with antibodies conjugated to oligonucleotide PEA probes at 4°C. After antibody binding, oligonucleotide annealing, and extension steps, the pre-amplification mix was added to the samples at room temperature. PCR amplification was performed, and PCR amplicons were then pooled and subjected to another PCR amplification step after addition of individual sample index sequences.

After pooling of the samples, bead purification and quality control of the generated libraries were performed. Sequencing was carried out using Illumina’s NovaSeq 6000 instrument. Quality control and normalization processes were then performed to translate barcode sequence counts into normalized protein expression (NPX) units.

Assays on the samples were performed blinded from the clinical data. Significant differences in individual protein levels (NPX units) between obese and non-obese was determined using T tests (two groups were compared) and adjusted for multiple comparisons by the Bonferroni and Hochberg method (R v4.2.3). Statistical comparisons of individual proteins between three or more groups were determined using one-way ANOVA (GraphPad Prism v9.5.0).

For bioinformatic analysis, differentially abundant serum proteins were mapped to biological pathways using Ingenuity Pathway Analysis (IPA) (QIAGEN Inc.)80.