General information:
B-cell precursor acute lymphoblastic leukemia (pre-B ALL) with mixed-lineage leukemia gene rearrangement (MLL-r) is a poor-prognosis subtype for which additional therapeutic targets are needed. To identify changes that could distinguish leukemic from normal precursor B cells, we performed integrated multi omic analyses on primary patient samples. MLL-r cells showed massive remodelling of their glycoprotein glycocalyx, with increased Core 2-type O-glycans and complex N-glycans as well as changes in sialylation and fucosylation. Shifts from chondroitin sulphate to heparan sulphate synthesis were also suggested. A survival screen to determine if glycan remodelling enzymes are redundant identified MGAT1 and NGLY1, components of the N-glycosylation/degradation pathway, as essential. OGT and OGA, unique enzymes that regulate intracellular O-GlcNAcylation were also indispensable. Transcriptomics and proteomics further identified the Fes tyrosine and GALNT7-mediated glycosylation as possible therapeutic targets. In addition to well-known MLL-r pre-B ALL glycoprotein markers, our integrated multi-omics workflow identified previously unrecognized diagnostic/therapeutic protein candidates.
Biologically derived material - Recombinantly produced material
Cell type:
N/A
Growth/harvest conditions for recombinantly produced material:
N/A
Biologically derived material - Biological origin of Material
Origin (biological fluid, tissue, etc):
tissue
Species:
Homo sapiens (Human)
Treatments and/or storage conditions:
Starting materials for isolation of control healthy precursor B cells were four BM samples (R7B1, R7B3, R7B6 and R7B11) from different donors depleted of CD34+ stem cells. R7B11 was not further processed, whereas R7B6 was enriched in CD19+ B cells, and R7B1 and R7B3 were enriched for both B and T-cells [CD19 and TCRα/β]. To isolate normal CD19+CD10+ precursor B-cells, around 2x10^9 of such viably frozen normal bone marrow cells from each sample were applied to EasySep™ Release Human CD19 Positive Selection Kit (STEMCELL Technologies, Vancouver, BC, Canada, Cat#17754) and EasySep™ Human CD10 Positive Selection Kit (STEMCELL Technologies, Cat#18358) columns.
MLL samples in this study were BM0 [(11q23) relapse sample, 98% blasts], BM37 [(46XY, t(9;11) diagnosis, 98% blasts], and BM41 (60% blasts, MLL rearranged (11q23; t9;11) 46XY]. Ficoll Plaque Plus (GE Healthcare, Cat#17-1440-02) centrifugation was used to remove red blood cells, according to manufacturer’s instructions and after washing viably, frozen with DMSO. Viably frozen cells were thawed and washed with twice with ice-cold DPBS (500xg, 4 min, 4°C). Each sample, consisting of 4-6x10^6 total cells, was divided into two fractions, one for proteomic/glycomic analyses and the other for RNA sequencing.
Cells were frozen -80°C until processed.
Glycoprotein:
N/A
Biologically derived material - Purchased from commercial manufacturer
Vendor and applicable item information:
N/A
Synthesis steps or specify where the equivalent reaction protocol is available:
N/A
Description of starting material:
N/A
Enzymatic treatments
Enzymes used for oligosaccharide removal or modification of starting material:
Release method-PNGaseF TREATMENT
Describe vendor or expression and purification procedure:
NEB
Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
37°C, 16 hours
Enzymatic treatments
Enzymes used for oligosaccharide removal or modification of starting material:
N/A
Describe vendor or expression and purification procedure:
N/A
Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
N/A
Chemical treatments
Define the technique for oligosaccharide release or other chemical modifications:
N/A
Reaction conditions (temperature, duration, volume and chemical concentrations):
N/A
Chemical treatments
Define the technique for oligosaccharide release or other chemical modifications:
Release method-REDUCTIVE BETA ELIMINATION
Reaction conditions (temperature, duration, volume and chemical concentrations):
50°C, 16 hours (O-linked glycan release)
Enzymatic modifications
Describe any treatments made to the isolated material:
N/A
Enzyme concentration, supplier, biological source, incubation time and temperature:
N/A
If novel glycosidase was used, provide information indicating the origin (i.e. species) of the enzyme:
N/A
Chemical modifications
Describe any treatments made to the isolated material:
N/A
Explain the type of modification employed:
N/A
Source of materials, description of kits used, reaction conditions and detailed workflow:
N/A
Purification steps:
N/A
Sample name:
N-linked oligosaccharides
N/A
Instrument manufacturer:
Bruker Daltonics
Instrument model:
Other
Customizations:
N/A
Ion mode:
Negative
Software name:
Compass
Version:
N/A
Upgrades not reflected in version number:
N/A
Switching criteria (tandem only):
N/A
Isolation width (global, or by MS level):
N/A
Location of ‘parameters’ file:
N/A
Supply type (static, or fed):
CaptiveSpray ion Source
Interface name:
CaptiveSpray ion Source
Catalog number, vendor, and any modifications made to the standard specification:
N/A
Sprayer name:
CaptiveSpray ion Source
Sprayer type, coating, manufacturer, model and catalog number (where available):
N/A
Relevant voltages where appropriate (tip, cone, acceleration):
N/A
Degree of prompt fragmentation evaluated:
N/A
Whether in-source dissociation performed:
N/A
Other parameters if discriminant for the experiment (such as nebulizing gas and pressure):
N/A
Plate composition (or type):
N/A
Matrix composition (if applicable):
N/A
Deposition technique:
N/A
Relevant voltages where appropriate:
N/A
Degree of prompt fragmentation evaluated:
N/A
PSD (or LID/ISD) summary, if performed:
N/A
Operation with or without delayed extraction:
N/A
Laser type (e.g., nitrogen) and wavelength (nm):
N/A
Other laser related parameters, if discriminating for the experiment:
N/A
Hardware options:
N/A
Collision-Induced Dissociation (CID)
Gas composition:
Ultra high purity Helium
Gas pressure:
N/A
Collision energy CID/function:
Collision induced dissociation (CID) MS/MS spectra were acquired for a m/z range between 50-2000, applying an isolation width of 2 Da and ICC target set to 40,000.
Electron Transfer Dissociation (ETD)
Reagent gas:
N/A
Pressure:
N/A
Reaction time:
N/A
Number of reagent atoms:
N/A
Electron Capture Dissociation (ECD)
Emitter type:
N/A
Voltage:
N/A
Current:
N/A
Reflectron status (on, off, none):
N/A
Final MS stage achieved:
N/A
Gas:
N/A
Pressure:
N/A
Instrument-specific parameters:
N/A
Peak selection:
N/A
Pulse:
N/A
Width:
N/A
Voltage:
N/A
Decay time:
N/A
IR:
N/A
Other parameters:
N/A
Detector type:
N/A
UniCarb-DR 
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