Autoflex maldi tof

The spots of interest were cut, reduced, alkylated, digested, and automatically transferred to a Matrix-assisted laser desorption/ionization (MALDI) analysis target by a Proteineer SP II and DP robot using the SPcontrol 3.1.48.0 v software (Bruker Daltonics, Bremen, Germany), with the aid of a DP Chemicals 96 gel digestion kit (Bruker Daltonics, Germany) and processed in a MALDI-TOF Autoflex (Bruker Daltonics, Germany) to obtain a mass fingerprint. We performed 100 satisfactory shots in 20 shotsteps, the peak resolution threshold was set at 1500, the signal/noise ratio of tolerance was 6, and contaminants were not excluded [5 (link)]. The spectrum was annotated by the Flex Analysis 1.2 v SD1 Patch 2 (Bruker Daltonics, Germany). The search engine MASCOT was used to compare the fingerprints against the UNIPROT [46 (link)] with the following parameters: Taxon-mouse, mass tolerance of up to 200 ppm, one miss-cleavage allowed, and as the fixed modification carbamidomethyl cysteine and oxidation of methionine as the variable modification.

PGLa (GMASK AGAIA GKIAK VALKA L-NH₂) and magainin 2a (GIGKF LHSAK KFGKA FVGEI MNS-NH_2; where the extension a is referring to the amidated carboxy terminus) were prepared by solid-phase synthesis using a Millipore 9050 automatic peptide synthesizer and Fmoc chemistry. The peptides were purified by reverse phase HPLC (Gilson, Villiers-le-bel, France) using a preparative C18 column (Luna, C-18-100Å-5 μm, Phenomenex, Le Pecq, France) and an acetonitrile/water gradient. Their identity and purity (>90%) were checked by MALDI-TOF mass spectrometry (MALDI-TOF Autoflex, Bruker Daltonics, Bremen Germany). The purified peptides were dissolved three times in 2 mM HCl at a 1 mg/mL concentration with subsequent lyophilization to ensure exchange of the TFA counter-ions. Quantification of peptides was based on mass and was determined for aliquots of ~10 mg peptide. Clean glass vials were weighted prior to the addition of the peptide solubilized in 2 mM HCl. The glass vials were weighted again after lyophilization. All vials were weighted 3 times before and after the addition of peptide on a 0.0001 g precision scale to account for small variations. Hereafter, the peptides were stored in aliquots of 1 mg at −20°C.

NCp7 and NCp8 proteins were purified as recombinant glutathione S-transferase (GST) fusion proteins from E. coli BL21—Codon Plus (DE3)—RIL cells (Stratagene), based on engineered expression vectors pGEX-4 T-3-NCp7 and pGEX-4 T-3-NCp8 [58 (link)]. The HIV-1_NL4–3 and HIV-2_ROD nucleocapsid protein coding sequencescoli BL21—Codon Plus (DE3)—RIL cells (Stratagene), based on engineered expression vectors pGEX-4 T-3-NCp7 and pGEX-4 T-3-NCp8 [58 (link)]. The nucleocapsid protein coding sequences of HIV-1_NL4–3 and HIV-2_ROD were taken from http://ncbi.nlm.nih.gov. GST-tagged proteins were purified by affinity chromatography on Glutathione-Sepharose (Amersham Pharmacia Biotech.) and the GST tag was cleaved off with thrombin enzyme. The proteins were further purified on a Superdex 200 FPLC column, and molecular masses were determined using MALDI TOF (Autoflex, Bruker Daltonics). The purified proteins were lyophilized and stored at −80°C. Synthetic (8–48) NCp8 peptide was obtained from ThermoFischer Scientific. Proteins were dissolved in freshly prepared, oxygen-free buffer containing 30 mM HEPES pH 6.5, 30 mM NaCl and 0.1 mM ZnCl₂. Activity of each protein preparation was compared with the activity of the synthetic full-length (48aa) NCp8 (Additional file 2: Figure S2).