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This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.
Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.
Protein glycosylation is a prevalent and complex type of co- and post-translational modification of proteins produced by species across the phylogenetic tree of life1,2,3. The protein-linked glycans are known to impact a wide span of biological processes important for human health, including mediation and regulation of cellular interactions and communication events4,5. Aberrant protein glycosylation has been thought to be a cause of malignant transformation, tumor progression, and spread6,....
The study was approved by the Human Research Ethics Committee (Medical Sciences) at Macquarie University, Sydney, Australia (Protocol 5201800073).
NOTE: The glycomics-guided glycoproteomics method can be applied to a diverse set of biological samples ranging from low to extreme glycoprotein complexity. For label-free glycoproteomics approaches (in which sample pooling is not performed), approximately 100-200 µg total protein from complex mixtures is required as starting material to ensure high glycoproteome coverage after enrichment for glycopeptides that may only constitute 1%-10% of the peptide population. Due to space constrain....
Representative results of the glycomics-guided glycoproteomics method applied to a FFPE tissue slide from a patient suffering from CRC in stage II are provided in this section.
To achieve sufficient protein starting material for the protocol, protein extracts from two slides (z stacks) were combined from the same FFPE tissue block following a published protocol and a TMT labeling approach was applied to increase the sensitivity of the glycoproteomics experiment (see optional steps below)
Critical steps in the protocol
In this protocol paper, we have outlined step-by-step the glycomics-guided glycoproteomics method, which provides a comprehensive view of the glycoproteome's heterogeneity and dynamics in the complex TME.
A critical step in the protocol is to ensure complete proteolysis without over-digesting the protein mixture. Non-specific cleavages of proteins inherently increase the peptide search space, leading to longer search times and a higher .......
The authors declare no conflict of interest.
THC is supported by an International Research Training Program Scholarship funded by the Australian Government. NB is supported by International Macquarie University Research Excellence Scholarships funded by Macquarie University. AC is supported by a Research Training Program scholarship funded by the Australian Government. RK was supported by the Cancer Institute of New South Wales (ECF181259). MTA is the recipient of an Australian Research Council Future Fellowship (FT210100455).
....Name | Company | Catalog Number | Comments |
Chemicals | |||
Ammonium acetate | Sigma Aldrich | A1542 | Alternatives available |
Ammonium bicarbonate, purity ≥99.0% | Sigma Aldrich | A6141 | Alternatives available |
Anhydrous acetonitrile (ACN), LC-MS grade | Sigma Aldrich | 34851 | Alternatives available |
Bovine fetuin | Sigma Aldrich | F3004 | Alternatives available |
Dithiothreitol (DTT) | Sigma Aldrich | D0632 | Alternatives available |
Ethanol | Sigma Aldrich | E7023 | Alternatives available |
Formic acid, LC-MS grade | Sigma Aldrich | 00940 | Alternatives available |
Glacial acetic acid | Sigma Aldrich | A6283 | Alternatives available |
Iodoacetamide (IAA) | Sigma Aldrich | I1149 | Alternatives available |
Methanol | Sigma Aldrich | M1775 | Alternatives available |
Peptide-N-glycosidase F (PNGase F) | Promega | V4831 | Elizabethkingia miricola PNGase F (10 U/μL) recombinantly expressed in Escherichia coli |
Polyvinylpyrrolidone 40 (PVP) | Sigma Aldrich | PVP40 | Alternatives available |
Potassium hydroxide (KOH) | Sigma Aldrich | 484016 | Alternatives available |
Sequencing-grade porcine trypsin | Promega | V5113 | Alternatives available |
Sodium borohydride (NaBH4) | Sigma Aldrich | 213462 | Alternatives available |
Triethylammonium bicarbonate (TEAB), LC-MS grade | Sigma Aldrich | T7408 | Alternatives available |
Trifluoroacetic acid (TFA), LC-MS grade | Sigma Aldrich | T6508 | Alternatives available |
Tools/Materials | |||
C18 disks | Empore | 66883-U | |
C8 disks | Empore | 66882-U | |
Flat-bottom polypropylene 96-well plate | Corning | 3364 | |
Immobilon-PSQ polyvinylidene difluoride (PVDF) membrane | Millipore | IPVH20200 | Pore size: 0.45 μm |
Microcentrifuge | Eppendorf | 5452000069 | Alternatives available |
Microcentrifuge adapters | The Nest Group, Inc., MA | SS18V | MiniSpin Column Collar, comes with Microspin Columns |
Oligo R3 resin | Thermo | 1133903 | Particle size 30 μm |
Parafilm | Parafilm M | PM996 | |
Protein LoBind tubes 1.5 mL | Eppendorf | 0030108450 | |
Protein LoBind tubes 2.0 mL | Eppendorf | 0030108442 | |
Safe-lock tubes 1.5 mL | Eppendorf | 0030120086 | |
Safe-lock tubes 2.0 mL | Eppendorf | 0030120094 | |
Shaker | Eppendorf | 5382000066 | Alternatives available |
Single hole puncher | Swingline | 74005 | |
SpeedVac concentrator | Martin Christ | RVC 2-25 CDplus | Alternatives available |
Supelclean ENVI-Carb SPE resin | Supelco | 57088 | Particle size 120-400 mesh. Resin can be manually extracted from cartridges, and can be stored long-term in 50% (v/v) methanol in MilliQ water |
Syringe 5 mL | Sigma Aldrich | Z116866 | Alternatives available |
Temperature-controlled incubator | Thermoline | E7.30 | Alternatives available |
Ultrasonic bath | Unisonics | FXP | Alternatives available |
ZIC-HILIC resin | Merck | 150455 | Particle/pore size, 5 μm/200 Å. Resin can be manually extracted from LC column, and can be stored long-term in 50% (v/v) methanol in MilliQ water |
ZipTip C18 solid-phase extraction (SPE) micro-columns | Millipore | ZTC18S096 | Alternatively, home-made C18-SPE micro-columns can replace commercial product |
LC-MS/MS Analysis | |||
1260 Infinity Capillary HPLC system | Agilent | Alternatives available | |
Analytical LC column pre-packed with ReproSil-Pur C18 AQ resin | Dr Maisch, Ammerbuch-Entringen, Germany | r13.aq.s2570 | Particle/pore size: 3 μm/120 Å, length/inner diameter: 25 cm/75 μm. Commercial C18 nano-LC columns also available/ |
Dionex UltiMate 3000 RSLCnano LC System | Thermo | Alternatives available | |
HyperCarb KAPPA PGC capillary LC column | Thermo | Discontinued | Particle/pore size, 3 μm/250 Å; column length, 30 mm; inner diameter, 0.180 mm. Larger geometries of the HyperCarb PGC-LC columns, producing similar quality data are commercially available (e.g., Thermo #35003-031030). SupelTMCarb LC column from Merck with a particle/pore size, 2.7 μm/200 and with different column lengths and internal diameter are also available (e.g., Merck #59994-U). |
LCMS-grade acetonitrile | LiChrosolv | 100029 | Alternatives available |
Linear trap quadrupole (LTQ) Velos Pro ion trap mass spectrometer (glycomics) | Thermo | Alternatives available | |
Orbitrap Fusion Lumos Tribrid Mass Spectrometer (glycoproteomics) | Thermo | Other high-resolution MS systems with or without ETD (only required for O-glycoproteomics) are also available (e.g., Q-Exactive HF-X Hybrid Quadrupole-Orbitrap, Thermo, TIMS-ToF-MS, Bruker or similar high performance mass spectrometers from other vendors) | |
Total Recovery Clear Glass screw vials 0.9 mL | Thermo | THC11093563 | Alternatives available |
Total Recovery Clear Glass screw vials matching caps | Thermo | THC09150869 | Alternatives available |
Trap LC column packed in-house with ReproSil-Pur C18 AQ resin | Dr Maisch, Ammerbuch-Entringen, Germany | r15.aq.s0202 | Particle/pore size: 5 μm/120 Å, length/inner diameter: 2 cm/100 μm. Commercial C18 trap LC columns also available. |
Software | |||
Byonic | Protein Metrics Inc | v2.6.46 or higher | Commercial glycopeptide and PTM search engine, accepting LC-MS/MS raw data from most MS vendors |
Byos | Protein Metrics Inc | v3.9-7 or higher | Commercial software for manual inspection of glycopeptide candidates and the automatic annotation of glycan MS/MS spectra |
GlycoMod | Expasy | Open access software, assisting the annotation of glycomics data (https://web.expasy.org/glycomod/) | |
GlycoWorkBench | EUROCarbDB | v2.1 | Open access software, assisting the annotation of glycomics data and the drawing of glycan cartoons |
RawMeat | VAST Scientific | v2.1 | Open access software, extracting m/z of glycan precursor ions from raw spectral data |
Skyline | Brendan X. MacLean | v21.2 or higher | Open access software for relative quantitation of glycans |
Xcalibur | Thermo | v2.2 or higher | For browsing raw LC-MS/MS data |
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