Systems biology of blood and tissue for comprehensive analysis of immune response to hepatitis B vaccine in adults

ABSTRACT: Protein concentrations of both lysed WBC and plasma samples were measured by the PierceTM Bradford Assay and normalized to contain 10 μg per sample before proceeding with sample digestion using the procedure as previously described44. Peptides were desalted using STop-And-Go Extraction tips (STAGE tips)45, dried using the Vacufuge Plus (Eppendorf) for 45 minutes, then chemically dimethylated with light, medium, and heavy formaldehyde46 for triplex analysis of each timepoint per individual (i.e. two triplex samples were prepared for each individual, with sample from one of the timepoints spiked into both triplexed samples to use a reference). After labeling, samples for each triplex was combined and desalted and dried again using STAGE tips and the Vacufuge Plus. Peptides were resuspended in 30μl of 0.1% formic acid for liquid chromatography and mass spectrometry analysis (LC-MS). A total of 2 μg per sample was injected into the EasynLC-1000 chromatography system (Thermo) with a 50 cm analytical column, packed in-house with C18, coupled to an Impact II Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany), with detailed parameters as previously described47. Data was analysed using MaxQuant (v1.5.5.1) with default values and “Match Between Runs” activated, searched against the human Uniprot database (downloaded on 15 July, 2017).

INSTRUMENT(S): impact II

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Primary Cell, Blood Plasma

SUBMITTER: Queenie Chan  

LAB HEAD: Leonard J Foster

PROVIDER: PXD020474 | Pride | 2020-12-01

REPOSITORIES: Pride