Ultraflex maldi tof mass spectrometer

The treatment procedure of the lead compounds was the same as for the total DNA quantification. The SDS-PAGE was carried out followed by Coomassie blue staining. The bands were taken and digested with trypsin for 12 h. Trichloroacetic acid (0.5%) was employed to acidify the digested proteins. The MALDI-mass analysis was carried out on a Bruker Ultraflex MALDI-TOF mass spectrometer. The spectra were collected from 400 shots per spectrum over an m/z range of 600 ∼ 3000, and calibrated by 4-point internal calibration. The fold change of the protein expression after lead compound treatment was estimated by band quantification of Prodigy Samespots analysis software. The protein masses were assigned and used for a database search with the MASCOT search engine¹. The detailed information of mass/mass analysis was the same as previous study (Pan et al., 2012 (link)).

Pull-down products containing eluted proteins were boiled, subjected to 8–16% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and visualized by silver staining or Western blotting. Each protein band was excised, destained, reduced, alkylated, and digested with trypsin. To extract the polypeptides, gel particles were subjected twice to consecutive 20 mM sodium bicarbonate and 5% formic acid in 50% acetonitrile treatments. The supernatants were combined and lyophilized, and the dried polypeptides were recovered by adding 10 μL of 0.1% formic acid, followed by sonication for 1 min. The recovered polypeptides were further purified using a ZipTip C18 column (Millipore, Billerica, MA), and eluted with acetonitrile to a final volume of 3 μL. Protein bands were excised and identified using in-gel trypsin digestion, then analyzed using a Bruker Ultraflex MALDI-TOF mass spectrometer (Bremen, Germany). After removing the masses derived from the standards, trypsin, matrix proteins, and keratins, the monoisotopic mass lists for each protonated peptide were subjected to database searches, and mass lists were exported to the Biotool 2.0 software package to perform peptide mass fingerprinting, using the Mascot (http://www.matrixscience.com) algorithm scoring to identify proteins.

Peptide fragments of wheat and barley AMP-like peptides were produced by solid-phase synthesis using Fmoc chemistry (Elabscience Biotechnology Inc., Wuhan, China). The synthesized peptides were purified by RP-HPLC. Their identity to the required sequences was proved by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis on an Ultraflex MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in a linear or reflector positive ion mode using α-cyano-4-hydroxycinnamic acid as a matrix.
The following characteristics of the synthesized peptides were calculated using the ExPASy ProtParam tool [41 ]: molecular weight, pI, net charge at pH 7, GRAVY index and aliphatic index. Hydrophobic moment μH was calculated with HeliQuest [42 (link)]. The Boman index was computed using APD3 [43 (link)]. The prediction of antimicrobial properties was carried out with CAMPR3 [44 (link)].