MassIVE MSV000090092

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Calibration of a mass spectrometry protocol for dental calculus protein analysis

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Description

Protein Processing: Following protein precipitation, samples underwent reduction by adding Dithiothreitol (Merck) to a final concentration of 10mM for 60 min at 55C, followed by alkylation by adding Iodoacetamide (Merck) to a final concentration of 18.75 mM for 30 minutes at room temperature. Subsequently, samples were digested overnight at 37C with 10 nG MS Grade Trypsin (Promega). Trypsinization was quenched with 1 uL of 0.1% Trifluoroacetic acid (TFA) (ThermoFisher). Solid Phase Extraction Stage Tips for Detergent Removal: Prior to mass spectrometry, samples were cleaned with a solid phase extraction method using 2 layers of C18 Empore SPE Disks (Merck) followed by 2 layers of SCX strong cation exchange Empore SPE Disks (Merck). Samples were then lyophilized . MS analysis: MS analysis was performed at the National Proteomics Unit at the Technion, Israel. The peptides were resolved by reverse-phase chromatography on 0.075 X 300-mm fused silica capillaries packed with Reprosil reversed phase material (Dr Maisch GmbH, Germany). The peptides were eluted using a linear 60 or 120 minutes gradient of 5 to 28%, a 15 minutes gradient of 28 to 95%, and 15 minutes at 95% Acetonitrile with 0.1% formic acid in water at flow rates of 0.15 u/min. MS analysis was performed by Q Exactive HF mass spectrometer (Thermo) in a positive mode (m/z 300-1800, resolution 120,000 for MS1 and 15,000 for MS2) using repetitively full MS scan followed by high collision dissociation (HCD, at 27 normalized collision energy) of the 20 most dominant ions (>=1 charges) selected from the first MS scan. The AGC settings were 3x10 6 for the full MS and 1x10 5 for the MS/MS scans. The intensity threshold for triggering MS/MS analysis was 1.3x10 5. A dynamic exclusion list was enabled with exclusion duration of 20s. [doi:10.25345/C5K06X51Z] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: dental calculus

Contact

Principal Investigators:
(in alphabetical order) 		Daniel Bar, Tel Aviv University, Israel
Submitting User: dbar
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