Isolation Of Formal Lab Report Essay
Isolation of exosomes from human serum
The initial volume of serum for the experiments was 4 mL per sample. The samples were diluted with an equal volume of PBS (AppliChem, St. Louis, MO) to decrease the viscosity. The diluted serum samples were centrifuged at 2,000×g for 10 min and 10,000×g for 30 min at 4 °C to remove dead cells and cell debris. The supernatant was transferred into Ultra-ClearTM tubes (Beckman Coulter, Indianapolis, IN) and centrifuged at 100,000×g using a Beckman Optima XL-70 Ultracentrifuge for 70 min at 4 °C. The supernatant was removed with a pipette and 2 mm of supernatant remained above the pellet. After one cycle of ultracentrifugation, it was not possible to remove all of the serum supernatant, where around 2 mm of …show more content…
The sample was cooled down and 25µL of 250mM of iodoacetamide was added, and then diluted with 1mL of 8M urea buffer (containing 50 mM TEAB). Next, the filter-aided sample preparation (FASP) method was performed. The sample solution was transferred to a centrifugal spin YM-30 filter (Millipore, Billerica, MA), and centrifuged at 14,000×g for 20 min. Then 200µL 8M Urea was added to wash the sample with the same centrifugation condition for 3 times. To remove the urea buffer, the sample was washed with 200µL of 50 mM TEAB also with this centrifugation condition for 3 times. The samples were then digested by 200ng sequencing grade modified trypsin (Promega, Fitchburg, WI) at 37 °C overnight. The tryptic digest was collected by centrifugation. The samples were acidified and desalted using a C18 tip. The eluted samples were dried by SpeedVac (Labconco, Kansas City, …show more content…
The samples were eluted under a 120 min linear gradient from 2% to 35% acetonitrile in 0.1% formic acid at a constant flow rate of 300nL/min.
An Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA) operated in positive ion mode was employed for sample analysis. The capillary temperature and the spray voltage were set as 200 °C and 2.5kV. The data was acquired in a data-dependent mode; the 15 strongest MS1 peaks were selected for subsequent MS2 analysis. For every selected peak, higher-energy collisional dissociation (HCD) was performed. The MS1 spectra (350-1650 m/z) and the MS2 spectra were both acquired in the Orbitrap.
The raw data were searched against the protein database by Proteome Discoverer 1.4 software (Thermo Fisher Scientific, San Jose, CA) with SEQUEST as the search engine. The parameters were set as follow: database, human UniProt; enzyme, trypsin; fixed modifications, carbamidomethyl (C) and 4-plex iTRAQ (N-term and K); variable modification, oxidation (M); up to 2 missed cleavages allowed; mass tolerance, 10 ppm for MS1 and mass tag, 0.05 Da for MS2; 1% false discovery rate allowed for
The initial volume of serum for the experiments was 4 mL per sample. The samples were diluted with an equal volume of PBS (AppliChem, St. Louis, MO) to decrease the viscosity. The diluted serum samples were centrifuged at 2,000×g for 10 min and 10,000×g for 30 min at 4 °C to remove dead cells and cell debris. The supernatant was transferred into Ultra-ClearTM tubes (Beckman Coulter, Indianapolis, IN) and centrifuged at 100,000×g using a Beckman Optima XL-70 Ultracentrifuge for 70 min at 4 °C. The supernatant was removed with a pipette and 2 mm of supernatant remained above the pellet. After one cycle of ultracentrifugation, it was not possible to remove all of the serum supernatant, where around 2 mm of …show more content…
The sample was cooled down and 25µL of 250mM of iodoacetamide was added, and then diluted with 1mL of 8M urea buffer (containing 50 mM TEAB). Next, the filter-aided sample preparation (FASP) method was performed. The sample solution was transferred to a centrifugal spin YM-30 filter (Millipore, Billerica, MA), and centrifuged at 14,000×g for 20 min. Then 200µL 8M Urea was added to wash the sample with the same centrifugation condition for 3 times. To remove the urea buffer, the sample was washed with 200µL of 50 mM TEAB also with this centrifugation condition for 3 times. The samples were then digested by 200ng sequencing grade modified trypsin (Promega, Fitchburg, WI) at 37 °C overnight. The tryptic digest was collected by centrifugation. The samples were acidified and desalted using a C18 tip. The eluted samples were dried by SpeedVac (Labconco, Kansas City, …show more content…
The samples were eluted under a 120 min linear gradient from 2% to 35% acetonitrile in 0.1% formic acid at a constant flow rate of 300nL/min.
An Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA) operated in positive ion mode was employed for sample analysis. The capillary temperature and the spray voltage were set as 200 °C and 2.5kV. The data was acquired in a data-dependent mode; the 15 strongest MS1 peaks were selected for subsequent MS2 analysis. For every selected peak, higher-energy collisional dissociation (HCD) was performed. The MS1 spectra (350-1650 m/z) and the MS2 spectra were both acquired in the Orbitrap.
The raw data were searched against the protein database by Proteome Discoverer 1.4 software (Thermo Fisher Scientific, San Jose, CA) with SEQUEST as the search engine. The parameters were set as follow: database, human UniProt; enzyme, trypsin; fixed modifications, carbamidomethyl (C) and 4-plex iTRAQ (N-term and K); variable modification, oxidation (M); up to 2 missed cleavages allowed; mass tolerance, 10 ppm for MS1 and mass tag, 0.05 Da for MS2; 1% false discovery rate allowed for