Fig. 6. Recombinant TREM-1 extracellular domain produced in human cells dimerizes in vitro.

a Native mass spectra of hTREM-1-ECD(21–200) at different concentrations. Signals representing monomeric and dimeric proteins are highlighted in orange and blue, respectively. (TREM-1-ECD(21–200) displays three predicted sites of N-glycosylation. Indeed, we observed that the high micro-heterogeneity of protein glycosylation resulted in overlapping signals from a wide distribution of glycoforms carrying different charges, making the charge states of TREM-1 ions unresolvable.) b MS signals of monomeric and dimeric hTREM-1-ECD(21–200): b, 1 native mass spectrum of 30 µM hTREM-1-ECD(21–200). Subpopulations of protein ions represented by the first and second peaks were mass-selected for subsequent collision induced dissociation (CID), releasing series of fragments. b, 2 and b, 3 show the resulting patterns of fragmentation derived from the corresponding mass selections. c Fractional mass of hTREM-1-ECD(21–200) dimer as a function of the protomer concentration of TREM-1. The dashed line indicates the level where half of TREM-1 molecules populates the dimeric state. d Fractional mass of hTREM-1-ECD(21–200) dimer as a function of the protomer concentration of TREM-1 (12 µM) in the presence of the LR12 peptide at different concentrations. The dashed line indicates the dimerization level in the absence of any peptide. The inset shows an exemplary mass spectrum of 12 µM TREM-1 in the presence of 18 µM LR12 peptide. e Analysis by CovalX HM4 system high-mass detector of hTREM-1-ECD(21–200) (0.250 mg/ml, 7.5 µM) without crosslinking (upper panel, in black) and after chemical crosslinking (lower panel, in red). Data information: data are representative of at least three independent experiments