Cbqca assay

Using a Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific), lysates were injected (200 μl per injection) onto a Superose 6 10/300 GL column (GE Healthcare) equilibrated with PBS (pH 7.2) with a flow rate of 0.2 ml min⁻¹. Twenty four fractions, each 200 μl in volume, were collected in a low protein binding 96-deep-well plate (Eppendorf). Approximate protein concentrations were estimated using the EZQ Protein Quantitation Kit (Thermo Fisher Scientific). Tris–HCl (1 M, pH 8.0) was added to each fraction to a final concentration of 0.1 M Tris–HCl to adjust the pH to 8.0. After reduction and alkylation using DTT and iodoacetamide, respectively, proteins in each fraction were digested to peptides for 18 h at 37 °C using either trypsin alone or both LysC & trypsin diluted in 0.1 M Tris–HCl (pH 8.0) at a final enzyme to protein ratio of 1:50 by weight. For peptide desalting, TFA was added to a 1% (v/v) final concentration, and peptides were purified using a Sep-Pak tC18 96-well μ-elution plate (Waters). Peptides were eluted with 500 μl of 50% (v/v) acetonitrile in 0.1% TFA, and dried in a SpeedVac prior to resuspension in 5% (v/v) formic acid. Peptide concentrations were determined using the CBQCA assay (Thermo Fisher Scientific) after 25-fold dilution of peptide samples in 0.1 M borate buffer (pH 9.3).

The EZQ protein assay and CBQCA assay were from Thermo Fisher Scientific (Waltham, MA). Triscarboxyethylphosphine (TCEP) (bond-breaker neutral pH solution) was from Pierce (Thermo Fisher Scientific). Trypsin MS-Grade was from Promega. Sep-Pak tC18 μ-elution 96-well plates were from Waters. The Pepmap C18 (2 cm × 75 μm) trap columns and EasySpray C18 columns (2 μm particles, 50 cm × 75 μm) were from Thermo Fisher Scientific. Complete protease inhibitor mixture tablets and PhosStop phosphatase inhibitor tablets were from Roche. All other materials were obtained from Sigma.

The size, polydispersity, and zeta potential of the NPs were determined by dynamic light scattering (DLS) by mixing 10 μL of a 25 mg/mL NP solution into 990 μL of MilliQ water using a Malvern Zetasizer ZSP. The release of encapsulated antigen from NPs was measured over 72 h as previously described (15 (link), 16 (link)). Briefly, 8 mg of NPs were dispersed in PBS and incubated at 37°C. At various timepoints NPs were centrifuged at 7,000 g and 4°C for 5 min and supernatant was collected and stored at 20°C for measurement using a Micro BCA assay (Pierce). Lyophilized NPs were washed by centrifugation with MilliQ water to remove cryoprotectant. Quantification of the total amount of antigen within NP was performed using the Micro BCA assay (Pierce) or CBQCA assay (ThermoFisher) as previously described by dissolving antigen-loaded NPs in 0.1M NaOH for 24 h (33 (link)).