14. (388) ASSAYS FOR GEL-BASED, SENSITIVE SCREENING OF REPRODUCIBLE DIFFERENCES IN CYTOSOLIC PROTEOMES BY COMPLEMENTARY, CELL-FREE REACTIONS LABELLING PROTEINS WITH NUCLEOTIDES. INFECTED MACROPHAGES AS MODEL

ASENSIO CRISTIAN JORGE ALEJANDRO; RODOLFO C GARCÍA

virtual

Congreso; REUNIÓN DE SOCIEDADES DE BIOCIENCIAS 2020; 2020

SAIC , SAI, SAFIS

AIMS: To optimize several conditions for novel, complementary biochemical assays allowing the screening of proteome differences between cell treatments by using cell-free, in vitro radiolabeling assays outperforming Coomassie and silver staining in sensitivity. METHODS: As model, macrophages were cultured and stimulated/ infected or not with different treatments, bacteria, lipopeptides and times. Sub-fractionated cytosols were incubated in parallel with different radiolabeled nucleotides and a large battery of combinatorial reaction components, additives and conditions. Cell-free reactions were separated in 1D and 2D gels comparing proteome profiles by staining versus radiolabeling, analyzing effects of reaction components and culture conditions. The sample size was 3-6 and 4-9 for each reaction and culture condition, respectively. After systematic iteration, we selected the reactions detecting infection time-dependent proteome differences with 90-100 % reproducibility and >20 % dysregulated level. RESULTS: The assays were versatile, robust and suited for cytosols, detecting different complementary proteome profiles depending on the in vitro labelling covalent linkage (phosphorylation, nucleotidylylation, ribosylation, AMPylation). Nucleotides labeled in gama or alpha phosphate worked differently. In vivo phosphorylations were evidenced by coupled phosphatase assays (in vivo sites were excluded from reactions unless dephosphorylated). Intentionally phosphorylating non-physiologic sites unoccupied in cells some proteins were labeled reproducibly, regardless of culture conditions, sensitively quantitating their expression level. Proteome radiolabeling outperformed staining in sensitivity for low-abundance proteins uncovering 5 novel, reproducibly altered proteins with time-dependent up-/down-regulation. CONCLUSION: the assays uncovered new markers and pathways of innate immune respons responses, one having a novel PTM and possible uses in vaccine/adjuvant testing and infectology.