Proteomics analysis comparing dsProsβ1- and dsmGFP-fed cabbage stem flea beetle adults.

Whole bodies of four CSFB adults receiving RNAi treatment were sampled on days 5, 15, and 30 post-emergence, frozen in liquid nitrogen, and homogenized by grinding. Proteins were extracted with a urea denaturation buffer (6 M urea, 2 M thiourea, 10 mM HEPES) at a ratio of 1 mL per 100 mg sample. Supernatants were recovered by centrifugation (12,000 g, 5 min), and protein concentrations were measured using a Bicinchoninic Acid (BCA) assay. Proteins were precipitated using a chloroform/methanol method, digested with RapiGest SF solution, treated with TFA (1:10), and incubated at 37°C for 45 minutes before drying in a speed vac. Samples were purified using a standard C18 stage tip protocol and analyzed by LC/MS at the Göttingen Center for Molecular Biosciences (Service Unit LCMS Protein Analytics). The proteomics experiments were repeated five times. The proteomics experiments included five biological replicates. Label-free quantification (LFQ) values were obtained by mapping peptides to the CSFB adult proteome. Proteins with fewer than three peptide counts were discarded. Statistical significance was set at adjusted P < 0.05, with significantly altered proteins defined by LogFold2 > 1 (increased) or LogFold2 < -1 (decreased). Proteins showing significant differential abundance were subjected to Gene Ontology (GO) enrichment analysis using the R package clusterProfiler (v3.17). The functional annotations of the proteins were retrieved from our previous work where Trinotate (v4.0, https://github.com/griffithlab/rnaseq_tutorial/wiki/Trinotate-Functional-Annotation) was used.