Sequencing grade modified trypsin

After SDS-PAGE, the band corresponding to the purified enzyme was excised from the gel, destained, and digested with sequencing-grade modified trypsin (Roche). Nano-LC-MS/MS analysis was conducted by using a Q-Exactive system (Thermo Scientific). The peptide mixture was separated using a nanoAcquity Ultra performance LC (Waters). A sample volume of 10 μL was loaded, and separation was performed at a flow rate of 200 nL min⁻¹ using 0.1% formic acid (v/v; solvent A) and 0.1% formic acid in acetonitrile (v/v; solvent B). The HPLC linear gradient for separation was 1–90% (v/v). A spray voltage of 2 kV was applied. The MS scan range was mass-to-charge ratio 300–1800, and the 10 most intense precursor ions were selected for subsequent MS/MS scans. The MASCOT Server 2.4 (Matrix Science) software was used for MASCOT database searching. Peptide data were searched against the NCBI non-redundant database (Nov. 19, 2013). The type of search was MS/MS Ion Search with 2+ as peptide charge and Viridiplantae (Green Plants) as taxonomy, and the instrument type was ESI-TRAP. Up to two missed cleavage was allowed, and mass tolerance for protein identification was 10 ppm for MS and 20 mmu for MS/MS. Peptides were considered identified if the MASCOT score was over the 95% confidence limit based on the “identity” score of each peptide.

All of the protein spots were analyzed by MALDI-TOF/TOF mass spectrometry. The protein spots were carefully excised from the gel, destained using destaining solution (50% methyl cyanide, 25 mM acid ammonium carbonate), and then digested for 13 h with sequencing grade modified trypsin (Roche, USA). Peptides from digested proteins were used for MALDI-TOF/TOF analysis. The MALDI-TOF mass spectrometry measurement was performed on a Bruker Ultraflex^III MALDI-TOF-MS instrument (Bruker Daltonics, Germany), operating in reflectron mode with 20 kV accelerating voltage and 23 kV reflecting voltage. A saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid was used as the matrix. A 1-μl volume of the matrix solution and the sample solution at a ratio of 1:1 was applied to the Score384 target well. The SNAP algorithm (S/N threshold: 5; quality factor threshold: 30) in FlexAnalysis 2.4 was used to select the 150 most prominent peaks in the mass range m/z 700–4000. The subsequent tandem mass spectrometry (MS/MS) analysis was performed in a data-dependent manner, and the 10 most abundant ions were subjected to high energy collision-induced dissociation analysis. The collision energy was set to 1 keV, and nitrogen was used as the collision gas.

All of the protein spots generated by 2-DE were analyzed by MALDI-TOF/TOF MS. The protein spots were carefully excised from the gel, destained using destaining solution (50% acetonitrile, 25 mM acid ammonium carbonate), and then digested for 13 h using sequencing-grade modified trypsin (Roche). Peptides from the digested proteins were used for MALDI-TOF/TOF analysis. MALDI-TOF MS was performed using an Ultraflex^III MALDI-TOF mass spectrometer (Bruker Daltonics) operating in reflectron mode with 20 kV accelerating voltage and 23 kV reflecting voltage. A saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid was used as the matrix. A 1-μL volume of a mixture of the matrix and sample solutions at a 1:1 ratio was applied to the Score384 target well. The SNAP algorithm (S/N threshold: 5; Quality Factor Threshold: 30) in FlexAnalysis v.2.4 was used to select the 150 most prominent peaks in the mass range m/z 700–4,000. The subsequent MS/MS analysis was performed in a data-dependent manner, and the 10 most abundant ions were subjected to high energy collision-induced dissociation analysis. The collision energy was set to 1 keV, and nitrogen was used as the collision gas.

Surface proteins of SEC purified EVs of A. perfoliata were removed through hydrolysis with trypsin as previously described [49 (link)]. Briefly, SEC purified EVs were diluted with PBS to a final concentration of 200 μg in 250 μL total volume. Sequencing grade modified trypsin (100 μg/mL; Roche, U.K) was added to the EVs obtained a final concentration of 50 μg/mL and incubated for 5 min at 37 °C. The treated EVs were then centrifuged for 1 h at 100,000× g at 4 °C. The resulting supernatant was stored at −20 °C prior to gel free mass spectrometry analysis.

Botulinum neurotoxin is highly toxic and requires appropriate safety measures. All neurotoxins were handled in a class 2 biosafety cabinet equipped with HEPA filters. Commercially purified BoNT/G complex toxin was purchased (Metabiologics, Madison, WI). Sequencing-grade modified trypsin at 0.5 mg/mL in 50 mM acetic acid and sequencing grade chymotrypsin at 1 μg/μL in 50 mM ammonium bicarbonate was purchased (Roche, Pleasanton, CA). All chemicals were from Sigma-Aldrich (St. Louis, MO) except where indicated.

Coomassie brilliant blue staining was performed on the scanned 2-D-DIGE gel, and then differential protein spots were found by position comparison, but it was difficult to detect proteins with low background expression. Therefore, a 2-DE gel prepared with 1 mg of internal standard protein was used for staining to show spots that could not be determined from the 2D-DIGE gel.
After 19 differential protein spots were excised from 2-D-DIGE gel, each spot was destained in destaining buffer (25 mM ammonium bicarbonate, 50% v/v acetonitrile). Destained spots were dehydrated by acetonitrile and spun-dry, and digested with sequencing grade modified trypsin (Roche) at 37℃ for 16 h. The matrix-assisted laser-desorption ionization (MALDI) mass spectra were produced on an Ultroflex II MALDI time-of-flight/time-of-flight mass spectrometer (MALDI-TOF/TOF MS) (Bruker Daltonics, Germany) with use of FlexAnalysis 2.4 software. After tryptic peptide masses were transferred to a BioTools 3.0 interface (Bruker Daltonics), peptide mass fingerprintings (PMFs) were searched against the NCBInr protein database (http://www.ncbi.nlm.nih.gov/; NCBInr 20,071,214; 5,742,110 sequences) by use of Mascot software2.2.03(http://www.matrixscience.com; Matrix Science, London,U.K.).