General information:
O-linked glycans released from Caco-2 cell line
Growth/harvest conditions for recombinantly produced material:
Human colorectal adenocarcinoma Caco-2 cells were grown in Dulbecco’s Modified Eagle medium (Gibco, Thermo Fisher Scientific) with 10 % FCS. Cells were subcultured at 80% confluency and maintained at 37 °C in a humidified incubator with 5 % CO2. At day 0, when cells reached full confluency, 2 mM butyrate was added to the appropriate cells (at 2 mM final concentration in the cell culture flask). For the treated cells, butyrate was added at each medium change. Cells were grown in three separate culture flasks (three biological replicates) for both butyrate treated and spontaneously differentiated group. On days 5, 7, 14, 21 and 24 cells were collected. in biological triplicates. For harvesting of the cells, medium was removed and adherent cells were washed twice with DPBS and trypsinized using 0.25 % trypsin- 1 mM EDTA. To stop the trypsin activity, medium (without FCS) in a ratio of 2:5 (trypsin:medium; v/v) was added and cells were pelleted at 300 x g for 5 min. Cells were resuspended in DPBS and counted and aliquoted to ~2.0 x 106 cells per replicate and washed twice with 1 mL DPBS for 3 minutes at 100 x g. The supernatant was removed and cell pellets stored at -20 °C until further use.
Treatments and/or storage conditions:
Cell pellets were stored at -20C after harvesting before performing analysis.
For commercial material, vendor and applicable item information:
NA
Generation of chemically derived material:
NA
Purification steps:
Cation exchange desalting; Carbon SPE
Sample name:
Oligosaccharides
Instrument: Thermo Fisher 1100 Series
Manufacturer url: NA
Injector: DioneX autosampler
Injector settings: NA
Manufacturer: In-house
Model: Hypercarb
Type of Chromatography: Reverse phase
Type of Material: Porous graphitized carbon
Column diameter: 75 um
Column length: 10 cm
Particle size: 3 um
Manufacturer url: NA
Temperature: 45°C
Solvent a: 10mM ammonium bicarbonate
Solvent b: 60% ACN, 10mM ammonium bicarbonate
Other solvent: NA
Flow rate: 0,6ul/min
Flow gradient: 1%-50% buffer B
Run time: 73min
Phase: Reverse
Date stamp: Tue Jun 28 UTC 2022
Instrument: Bruker Daltonik amaZon Speed ETD ETD
Customizations from the manufacturer's specification:
NA
ion_mode: -
Sprayer type : Static
Interface name: NA
Other data for the Interface: Bruker Daltonik
Sprayer name: Captive spray
Other data for the sprayer: NA
Relevant voltages: 1000V
Degree of prompt fragmentation evaluated: Yes
In-source dissociation performed: Yes
Other parameters if discriminant for the experiment: methanol enriched nebulizing gas (N2)- nanobooster with dry gas temperature 280°C , 3 L/min and pressure 3 psi
Plate composition (or type): NA
Matrix composition: NA
Deposition technique: NA
Relevant voltages: NA
Degree of prompt fragmentation evaluated: NA
PSD (or LID/ISD) summary: NA
Operation with or without delayed extraction: NA
Laser type: NA
Other laser related parameters: NA
url: NA
Hardware options: Amazon speed CID
CID. Gas composition: Helium
CID. Gas pressure: NA
CID. Collision energy: Smart Frag with amplitude 29-120% within 32 ms with a default cut off
ETD. Reagent gas: NA
ETD. Pressure: NA
ETD. Reaction time: NA
ETD. Reagent atoms: NA
ECD. Emitter type: NA
ECD. Voltage: NA
ECD. Current: NA
TOF. Reflectron status: NA
Ion trap. Final MS stage achieved: NA
Ion mobility. Gas type : NA
Ion mobility. Pressure: NA
Ion mobility. Instrument parameters: NA
Peak selection: NA
FT-ICR. Pulse: NA
FT-ICR. Width: NA
FT-ICR. IR: Decay time: NA
FT-ICR. IR: NA
FT-ICR. Other parameters: NA
Detector type: NA
UniCarb-DR 
Supported by SIB ExPASy | JST / NBDCDICP
