Computational protocol: Profiling plasma extracellular vesicle by pluronic block copolymer based enrichment method unveils features associated with breast cancer aggression, metastasis and invasion

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Protocol publication

[…] In-solution trypsin digestion was performed according to manufacturer’s instructions (ThermoFisher). Samples were analysed by nanoflow reverse phase liquid chromatography using a Dionex Ultimate 3000 RSLCnano System (ThermoFisher) coupled in-line to a Q Exactive HF mass spectrometer (ThermoFisher). The nano-LC system included an Acclaim PepMap 100 C18 5 μm 100A 300 μm × 5 mm trap column and an EASY-Spray C18 2 μm 100A 50 μm × 150 mm analytical column (ThermoFisher). Peptide samples were eluted with a two-step gradient of 2–30% B for 28 min and then 30–45% B for 5 min, where B consisted of acetonitrile containing 0.1% formic acid. Blank samples consisting of 0.1% formic acid in water were injected between each sample and eluted with the same gradient profile and times as the samples. The LC system was interfaced with the MS using an EASY-Spray electrospray ion source (ThermoFisher) and the samples were analysed using positive ion spray voltage set to 2 kV, S-lens RF level at 65, and heated capillary at 285°C. The Q Exactive HF was operated in the data-dependent acquisition mode for fragmentation. MS1 survey scans (m/z 400–1400) were acquired in the Orbitrap analyser with a resolution of 120,000 at m/z 200, an accumulation target of 3 × 106, and maximum fill time of 50 ms. MS2 scans were collected using a resolution of 30,000 at m/z 200, an accumulation target of 1 × 105, and maximum fill time of 100 ms, with an isolation window of 1.5 m/z, normalized collision energy of 28, and charged state recognition between 2 and 7.ProteoWizard [] was used for peak-picking, filtering out peaks with intensity less than 100 and converting the file to mzML format. Protein search and identifications were performed using MS-GF+ [] search engine on Homo sapiens (Uniprot TaxID = 9606). For semi-quantification analysis, algorithm was establish according to the previous normalized spectra index algorithm [,] with modifications: sum of intensity from each peptide MS2 scan was normalized by the parent peptide charge, such normalized intensity for the same peptide from different MS2 scans was then sum together; sum of the normalized intensity for the same peptide is then further normalized by the experimental molecular weight of the peptide, as a unit of normalized intensity per Dalton (mass). Normalized intensity per mass between different peptides of the same protein detected in the same sample was then averaged to obtain the final spectrum index (SI). For relative abundance, the SI was further normalized by the average total MS2 intensity among all samples. […]

Pipeline specifications

Software tools ProteoWizard, MS-GF+
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms