*2.8. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI TOF MS) Analysis*

The molecule weight of OVM was determined by MALDI-TOF MS (4800 Plus MALDI-TOF/TOF Analyzer, AB Science, Framingham, MA, USA) according to the method of Liu et al. [21]. OVM samples were dissolved in distilled water at 1:100. The matrix solution was prepared by the mixing of sinapic acid (5 mg/mL) in 50% acetonitrile with 0.1% TFA. The OVM solution was mixed with the matrix solution at a ratio of 1:1. The mixtures (2.0 μL) were then dropped onto the MALDI target plate and allowed to dry at room temperature before analysis.

#### *2.9. High Performance Liquid Chromatography Orbitrap Tandem Mass Spectrometry (HPLC Orbitrap MS/MS) Analysis*

The OVM samples were digested by trypsin at 37 ◦C for 24 h. The digested sample solution was separated via a nanoliter flow HPLC system (UltiMate 3000RSLCnano, Thermo Fisher Scientific, Waltham, MA, USA). Solution A was an aqueous solution with 0.1% formic acid. Solution B was acetonitrile binary solution with 0.1% formic acid and 84% acetonitrile. The digested sample solution was injected into a RP-C18 column to remove insoluble or impure substances and then separated by another RP-C18 column at a flow speed of 300 μL/min. Gradient elution was then carried out.

After separation, the eluant was injected into an LTQ-Orbitrap Fusion Velos mass spectrometer (Thermo Fisher Scientific; Waltham, MA, USA) for analysis by tandem mass spectrometry (MS/MS) to identify protein modification forms and sites with a positive ion detection mode. The precursor ions were subjected to high-energy collisional dissociation (HCD) fragmentation to detect fragment ions. Twenty fragment maps (MS/MS2 scans) showing the mass-to-charge ratio of the polypeptide and polypeptide fragments were collected at each full scan. Raw files were obtained from the corresponding database using Proteome Discoverer 1.4. Some parameters were set as follows: Enzymes, nonspecified; Missed cleavage, 2; Modification, carbamidomethyl, oxidation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, nitro.

#### *2.10. Statistical Analysis*

The values were expressed as means ± standard deviation from three separate experiments. The analysis was performed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Statistical data were determined based on a two-tailed *t*-test using standard deviations.
