Hct ultra etdii ion trap mass spectrometer

According to the Jian study [36 (link)], an HCT Ultra ETDII Ion-trap Mass Spectrometer (Bruker Daltonics) interfaced with an UltiMate 3000 nano high-performance liquid chromatography system (Dionex) with a 15 cm by 75 μm C18 column was used. The sample’s peptides were eluted by means of an acetonitrile gradient at a flow rate of 0.3 mL/min.
The mass spectra for the eluted fractions were acquired as successive sets of scan modes. The MS scan gained the intensity of ions in the range of 200 to 2000 m/z, and a specific ion was selected for a tandem MS/MS scan. The centroid MS/MS data of enzyme-digested fragments were collected using HyStar 3.2, Bio-Tools, and WarpLC software (Bruker Daltonics). Then, the data were submitted to a search program (MASCOT) for searching Swiss-Prot databases of Homo Sapiens with the following settings: A mass tolerance of 0.3 Da for precursor and fragment ions, carbamidomethyl cysteine residues as fixed modifications, one missed cleavage acceptable for trypsin digestion, and oxidized methionine residues for an optional improvement.

An HCT Ultra ETDII Ion-trap Mass Spectrometer (Bruker Daltonics) interfaced with an UltiMate 3000 nano high performance liquid chromatography system (Dionex) with a 15 cm × 75 μm C18 column was used per the Jian study [33 (link)]. In brief, the peptides of the sample were eluted using an acetonitrile gradient at a flow rate of 0.3 mL/min. The spectra for the eluted fractions were acquired as successive sets of scan modes. The MS scan obtained the intensity of ions in the range of 200 to 2000 m/z, and a specific ion was selected for a tandem MS/MS scan. The centroid MS/MS data of enzyme-digested fragments were obtained using HyStar 3.2, Bio-Tools, and WarpLC software (Bruker Daltonics). Subsequently, the data were submitted to a bioinformatics search program (MASCOT) for searching Swiss-Prot databases based on Homo Sapiens with the following settings: a mass tolerance of 0.3 Da for precursor and fragment ions, one missed cleavage acceptable for trypsin digestion, carbamidomethyl cysteine residues as fixed modifications, and oxidized methionine residues for an optional modification.

Aliquots (2 mL) of growth medium collected from cultures of P. gingivalis strains ATCC 33277, ∆ppad (ATCC 33277), W83 and Δppad (W83) were lyophilized in an Alpha 1-2 lyophilizer (Martin Christ, Osterode am Harz, Germany) and then dissolved in 100 µL of ultra-pure water to achieve a 20-fold concentration. Three biological replicates were prepared. Samples for electrophoresis were prepared by mixing 15 μL of concentrated supernatant with 15 μL of loading buffer for subsequent SDS-PAGE electrophoresis followed by incubation at 95 °C for 5 min. After electrophoretic separation using the Laemmli system [80 (link)], protein detection was carried out with Coomassie Brilliant Blue G-250 staining and specific protein bands were excised from the gel. These gel samples were subjected to tryptic digestion followed by protein identification with LC-MS/MS using the Dionex UltiMate 3000 UHPLC system (Dionex, Carlsbad, CA) and the HCTUltra ETDII ion-trap mass spectrometer equipped with an electrospray ionization ion source (Bruker, Bremen, Germany), as described previously [81 (link)]. Protein identification was then performed using a SwissProt protein database search (560,118 sequences for all entries, including 336,487 sequences for bacterial proteins) with an in-house Mascot server (v.2.3.0, Matrix Science, London, UK).