PDE3A and PKG-Iα constructs were transiently transfected in HEK293 cells for 48 h. PDE3A was immunoprecipitated from the cell lysates as described previously. The protein was resolved using 4 to 20% Tris–glycine SDS-PAGE and visualized by colloidal Coomassie stain (Bio-Rad Laboratories). The band corresponding to PDE3A (approximately 130 kDa) was excised and destained with 100 mM ammonium bicarbonate/50% (v/v) acetonitrile. Protein in gel was subjected to reduction and alkylation of cysteine residues before overnight in-gel digestion with chymotrypsin in 100 mM Tris–HCl containing 10 mM CaCl2. The peptides were extracted with 0.1% TFA, 60% acetonitrile, and evaporated to near dryness. MS analysis of PDE3A was then performed as described (59 (link)). All spectra were taken on an ABSciex 5800 MALDI-TOF mass spectrometer in positive reflector mode (10 kV) with a matrix of cyano-4-hydroxycinnamic acid. At least 1000 laser shots were averaged to get each spectrum. The masses were calibrated to known peptide standards. The MS spectra were analyzed in the ProteinPilot software package (AB Sciex).
5800 maldi tof mass spectrometer
Manufactured by AB Sciex
Sourced in United States
The 5800 MALDI-TOF mass spectrometer is a laboratory instrument designed for the analysis of biomolecules. It utilizes matrix-assisted laser desorption/ionization (MALDI) technology coupled with time-of-flight (TOF) mass analysis to provide high-resolution mass spectra of samples. The core function of this equipment is to accurately determine the molecular masses of various analytes, such as proteins, peptides, and small molecules.
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3 protocols using 5800 maldi tof mass spectrometer
1
PDE3A Immunoprecipitation and Proteomic Analysis
2
In Vitro FGF21 Digestion by FAP
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For FAP in vitro digestion of FGF21, human or mouse FGF21 was incubated at 37°C with recombinant FAP (R&D Systems) at a ratio of 10:1 (FGF21:FAP) in digestion buffer (25 mM Tris, 0.25 M NaCl, pH 8.0). Aliquots were taken at different time points and analysed with an AB Sciex 5800 MALDI TOF mass spectrometer (AB Sciex) after desalting using C18 Ziptips.
3
Isolation and Identification of Antifungal Lipopeptides
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The antifungal substances contained in the crude extract were isolated by HPLC using a 1260 series instrument (Agilent, Santa Clara, CA, United States) equipped with a C18 reversed-phase column (150 mm × 4.6 mm, 5 μm). After consulting the literature and exploring the separation conditions, the optimum separation conditions were determined as follows: The mobile phase comprised (A) 0.1% trifluoroacetic acid in water and (B) acetonitrile (60:40), a flow rate of 0.8 ml/min, the column temperature was 25°C, and a UV detector was employed at a detection wavelength of 230 nm. The eluent of each peak was collected in an Eppendorf tube and the corresponding retention time was recorded. The eluents were vacuum dried and dissolved in sterile water for subsequent activity detection. Eluents possessing antifungal activity against Ggt were analyzed using a 5800 MALDI-TOF mass spectrometer (AB SCIEX, Redwood, WA, United States) employed in positive reflectron mode with a matrix comprising α-cyano-4-hydroxycinnamic acid (Velho et al., 2011 (link)). Isolated and identified lipopeptides from the culture supernatant of strain Z-14 were assessed by HPLC to determine their purity using the parameter settings described above.
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