Quantitative LC/MS/MS was performed on 2 uL (1 ug) of each sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipped with a FAIMSPro device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (-40v, -60v, -80v). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r 120,000 (m/z 200) full MS scan from m/z 375 - 1500 with a target AGC value of 4e5 ions was performed. MS/MS scans were acquired in the Orbitrap at r 50,000 ( m/z 200) from m/z 100 with a target AGC value of 1e5 and max fill time of 35 ms. The total cycle time for each CV was 1s, with total cycle times of 3 sec between like full MS scans. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hours.
[doi:10.25345/C5FN11345]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: extracellular vesicles, label free, proteomics
Principal Investigators: (in alphabetical order) |
Xin Zhang, Duke University, USA |
Submitting User: | es3064 |
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