Ltq ot

Label-free quantitation was performed by peptide mass spectrometry to determine the relative abundance of each HA present in the mosaic nanoparticle samples. Each mosaic nanoparticle, either before or after SEC purification, along with a standard mixture of each purified HA-I53_dn5B fusion protein at equimolar concentrations (1:1:1:1), was denatured and reduced using guanidine hydrochloride and DTT. Samples were then alkylated with iodoacetamide, deglycosylated with N-glycanase (New England Biolabs), and digested overnight with LysC protease (ThermoFisher scientific). LC-MS was performed using a Waters Acquity UPLC coupled to a Thermo LTQ-OT using data-dependent acquisition. Peptides were resolved over a Waters CSH C18 1.7 μm, 2.1 × 100 mm column with a linear gradient from 3% to 40% B over 30 minutes (A: 0.1% formic acid; B: acetonitrile with 0.1% formic acid). Peptides were identified from MS/MS data using Protein Prospector using a score cutoff of 15 (http://prospector.ucsf.edu/). Due to the high sequence identity between the HA constructs, only four peptides unique to each specific HA were observed that could be used for label-free quantitation. The integrated peak areas for these peptides relative to the areas from an equimolar mixture of each HA were used to estimate the total abundance of each HA within the mosaic nanoparticle samples (Supplementary Table 3).