Mass-spectrometry (MS) is a powerful method for high-throughput and robust analysis of cellular proteins and metabolites. Recent advances in native MS allow to have shown that it is possible to study cellular proteins directly from cell growth medium under native conditions and thus to get a deeper insight into cellular interactome processes [1]. Moreover, coupling the MS-based native proteomic analysis to 13C-tracer LC-MS/MS metabolic studies gives an opportunity to screen the effects of cellular metabolome alterations on the protein interactions.

Here we present a combined proteomic-metabolic MS-based investigation of the inactivation of Natural Killer cells in tumor microenvironment.

Recombinant human GrB was expressed in the yeast Pichia Pastoris. Upon expression and secretion of the protein, the cell growth media was collected and cleared with centrifugation. The resulting supernatant was buffer-exchanged into MS-compatible buffer. Each of the prepared samples was supplemented with heparan sulfate/serglycin in the estimated cellular concentration. The effect of different glycolytic compounds on the formation and stability of GrB - heparan sulfate/GrB - serglycin complexes was investigated on a commercial MS instrument (Synapt G2, Waters).

For metabolomic analysis Natural Killer cells were co-cultured with HCT116 human colon carcinoma cells in the presence of the 13C -glucose medium. After the co-culturing, cells were harvested and cellular metabolites were analysed both from the cells and from the medium. Targeted metabolomic analysis was performed using a high-resolution LC-MS/MS method [2]. Data analysis for 13C-labelled metabolite profiling was done using the XCalibur software.

Preliminary results of the metabolic profiling reveal that the glucose metabolism of Natural Killer cells undergoes substantial changes in the tumor microenvironment. The protein analysis shows that these metabolic alterations affect the formation of GrB - heparan sulfate/GrB - serglycin complexes responsible for the antitumor activity of the Natural Killer cells.

[1] Shay Vimer, Gili Ben-Nissan, Michal Sharon, Nature protocols, 2020, 15, 236-265.
[2] Andrew Palmer, Prasad Phapale, Ilya Chernyavsky et al. FDR-controlled metabolite annotation for high-resolution imaging mass spectrometry, Nat Methods, 2017, 14, 57–60.