Figure 1. Mass spectrometry (MS) analysis of lipo-NmMetQ and NmMetQC20A proteins.

(A) (Top) Schematic of lipoprotein maturation pathway. Inset contains a schematic of a lipoprotein with acyl chain composition [16:0,16:0,16:0]. Acyl chains are grouped in a dotted line box and their average masses are calculated. Below the schematic are the theoretical masses for the lipo-NmMetQ proteins (in italics) assuming triacylation occurs via the canonical lipoprotein maturation pathway due to the sequential action of three enzymes (Lgt, LspA, and Lnt). The numbers in the brackets correspond to the total number of carbons and double bonds, respectively, present in the fatty acyl chains of the lipid. (Bottom) Schematic of various NmMetQC20A proteins with example theoretical average masses, shown in italics, assuming cleavage occurs between A19 and A20, possibly by signal peptidase I (SPase I). N-terminal signal peptides are represented by a green rectangle. (B) Characterization of lipo-NmMetQ. Size-exclusion chromatogram and mass spectra of peak 1. The molecular masses of the major species correspond within 1 Da to the predicted mass for two triacylated NmMetQ species, one with acyl chain composition [16:0, 16:0, 16:0] (31,661 Da) and the other with [16:0, 16:0, 18:1] (31,685 Da). (C) Characterization of NmMetQC20A. Size-exclusion chromatogram and mass spectra of the major species from peak 2 and peak 3. The molecular masses of the major species of peak 2 and 3 correspond to the pre-protein NmMetQ (32,802 Da) and secreted NmMetQ (30,839 Da), respectively. These measured masses are within 3 Da of the predicted masses for each species. Assigned NmMetQ species are depicted in cartoon form on the chromatograms.

Figure 1—figure supplement 1. DLS and SEC-MALS measurements of NmMetQ proteins.

DLS and SEC-MALS measurements of NmMetQ proteins. (A) Representative DLS intensity distribution plots of lipo-NmMetQ (0.7 mg/ml), pre-protein (2.3 mg/ml) and secreted NmMetQ (2.7 mg/ml). The hydrodynamic radius (R_h), the polydispersity (Pd %), and molecular weight estimate based on the hydrodynamic radius of a folded globular protein (Mw-R) are listed below each plot. The mean and SEM of each measurement were calculated from triplicate measurements. (B) SEC-MALS profiles of NmMetNI (0.3 mg/ml, black trace), lipo-NmMetQ (0.05 mg/ml, green trace), pre-protein (0.1 mg/ml, yellow trace), and secreted NmMetQ (0.05 mg/ml, cyan trace). The UV absorbance at 280 nm (left y axis) is plotted against the SEC elution volume and overlaid with the molar mass determined for each peak (right y axis). The molar mass determined for each peak is indicated. Protein concentrations for the SEC-MALS experiments were determined at the highest point of the peak using UV as the concentration source and the appropriate extinction coefficient. We propose that both lipo-NmMetQ and pre-protein NmMetQ aggregate with DDM to form micelle-like complexes that depend on the experiment condition.