Spots were excised from gels, reduced, alkylated and digested with trypsin sequencing grade (Roche Molecular Biochemicals) [111 (link)]. Produced peptides were analyzed in a 4800 Plus Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, MDS Sciex, Toronto, Canada) at the Proteomics Unit of Complutense University of Madrid. The MS data was searched against SwissProt Data Base with taxonomy restriction to human (553,231 sequences) using MASCOT 2.3 (http://www.matrixscience.com ) search engine through Global Protein Server v 3.6 software (ABSciex). The search parameters were carbamidomethyl cysteine as fixed modification and oxidized methionine as variable modification. Peptide mass tolerance was 50 ppm and up to 1 missed trypsin cleavage site allowed. All identified protein outperformed the probability scores fixed by mascot as significant with a p-value minor than 0.05.
4800 plus proteomics analyzer maldi tof tof mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in Canada
The 4800 Plus Proteomics Analyzer is a MALDI-TOF/TOF mass spectrometer designed for high-throughput protein identification and characterization. It provides accurate mass measurements and tandem MS/MS capabilities for proteomic applications.
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3 protocols using 4800 plus proteomics analyzer maldi tof tof mass spectrometer
1
Mass Spectrometry-based Protein Identification
2
Recombinant Expression and Purification of TdLTP2
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Specific encoding cDNA of T. durum TdLTP2 (Gene Bank accession no. MK570866) was cloned via yeast expression vector pPICZαA and sequenced. Recombinant rTdLTP2 was expressed as non-tagged protein and purified as described elsewhere [32 (link)] with slight modifications. The RP-HPLC was used to purify the fraction obtained from gel filtration chromatography using the column. The elution of protein was performed at the flow rate of 1 ml/min, using a gradient of acetonitrile from 0 to 85% in 145 min, where the absorbance was measured at 214 nm. The purified intact rTdLTP2 was quantified by BCA Protein Assay Reagent (ThermoScientific) then analyzed by MALDI-TOF (UltraFlex II, Bruker Daltonics), and confirmed by Peptide mass fingerprinting in the Proteomics Unit of the Universidad Complutense de Madrid. Analysis was performed in a 4800 Plus Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, MDS Sciex, Toronto, Canada) using α-cyano-4-hydroxy-cinnamic acid matrix. Electrophoresis gel (SDS-PAGE) was carried out and the resolved proteins were electro-transferred onto a nitrocellulose membrane to visualize the protein.
3
Protein Identification and Characterization
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Protein identification and characterization were performed after their separation by SDS‐PAGE (15%). Protein bands were stained with PageBlue Protein Staining Solution (Fermentas International), and spots of interest were cut out from the gels with a sterile scalpel and sent for analysis to the Proteomic Service of the Complutense University of Madrid (UCM, Spain). There, the samples were reduced, alkylated and digested with trypsin, according to Sechi 2002.31 Afterwards, the supernatant was collected and placed on a matrix‐assisted laser desorption/ionization (MALDI) plate with a matrix of 3 mg/ml of α‐cyano‐4‐hydroxycinnamic acid (Sigma) in 50% acetonitrile. Analyses were performed in a 4800 Plus Proteomics Analyzer MALDI‐TOF/TOF mass spectrometer (Applied Biosystems) operated in positive reflector mode with an accelerating voltage of 20,000 V. In addition, all mass spectra were calibrated internally using peptides from the auto digestion of trypsin. For protein identification, the SwissProt database was searched with the Human taxonomy restriction (20,370 sequences; 11,359,082 residues) using MASCOT 2.6 (http://www.matrixscience.com ) via the Global Protein Server v3.6 software (ABSciex).
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