Mass spectrometry analysis of EV71 VLPs

For the peptide mapping and N- and C-terminus sequencing by liquid chromatography-mass spectrometry, the purified EV71 VLPs samples were reduced with 10 mM dithiothretiol (DTT) in 100 mM NH₄HCO₃, alkylated with 50 mM indoleacetic acid (IAA) in 100 mM NH₄HCO₃ and divided into three parts. The three samples were digested for 12–16 h at 37°C with a solution of trypsin, chymotrypsin or endoproteinase Glu-C (protease-to-substrate ratio was 1:50). To identify the N-glycosylation sites of EV71 VLPs, a solution of PNGase-F (protease-to-substrate ratio was 1:20) was adopted to digest the purified VLPs samples at 37°C for 12–16 h. Thereafter, the molecular weights of the peptide fragments and their amino acid sequences were measured by tandem mass spectrometry (LC-MS/MS) by using Q Exactive (Thermo Fisher Scientific, Waltham, MA, USA). The HPLC system consisted of an Eksigent Nano2D LC (Eksigent Technologies, Silicon Valley, CA, USA) and an Acclaim^® PepMap300 C18 (15 cm long, 75 μm inner diameter, 5 μm, 300Å) (Dionex, Sunnyvale, CA, USA). Electrospray MS/MS spectra were allocated to the EV71 VLPs primary sequence using Mascot 2.1.0 (Matrix Science, London, UK) software, and an in-house protein sequence database was established. The mass tolerance on the parent and the fragment ions were 0.1 Da and 0.2 Da, respectively. The variable modifications allowed were carbamidomethyl (C), oxidation (M) and deamination (D and E). The N-glycosylation sites were determined by the quality drift (0.984 Da) of asparagine changed to aspartic acid [25].

To identify the molecular weights of VP0, VP1 and VP3 capsid proteins of EV71 VLPs, the purified EV71 VLPs were mixed (1:1) with 5 mg/mL alpha-cyano-4-hydroxy-cinnamic acid (CHCA) containing 0.1% trifluoroacetic acid (TFA) and 50% acetonitrile. After mixing, 0.8 μL of the mixture was used for MS analysis, and then a MALDI-TOF/TOF mass spectrometer 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA) was adopted. Data Explorer (TM) software (Applied Biosystems) was used to analyze the data.