Name: Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments (Video) | JoVE Journal
Uploaded: 2025-02-07T12:30:00.000+00:00
Duration: 10 min 59 s
Description: Learn glycomics-guided glycoproteomics for comprehensive tumor microenvironment profiling using mass spectrometry. This method enables precise glycopeptide analysis and reveals cancer glycobiology insights.

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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).

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

The authors declare no conflict of interest.

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&#39;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 ...

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 spread6,7,8. This implicates glycans in key tumorigenic processes in the tumor microenvironment (TME) and offers a considerable and largely untapped potential for the discovery of glycan-based biomarkers and therapeutic applications. Glycobiology is, therefore, receiving increasing attention in cancer research and across life science9.
Powered by key developments in glycoanalytics and informatics over the past decade10,11,12,13,14,15,16,17, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-driven glycomics and glycoproteomics have been established as powerful tools for the system-wide profiling of liberated glycans and intact glycopeptides from complex mixtures of glycoproteins extracted directly from their biological sample origins such as various types of cancer patient specimens (e.g., tumor tissues, cell populations, bodily fluids)3,13,18,19,20.
Glycomics provides information on the structure and quantity of the glycan repertoire (the glycome), which is dynamically produced by cells, tissues, and even entire organisms. On the other hand, glycoproteomics provides quantitative information about the protein carriers and their site(s) of glycosylation, the site occupancy rates (macro-heterogeneity), the site-specific glycan compositions and their site distribution pattern (micro-heterogeneity) with limited glycan structural information (often limited to glycan composition)15,16,21. Hence, glycomics and glycoproteomics are complementary approaches to survey the biochemical details of the glycoproteome22.
A myriad of methodological designs and analytical strategies for glycomics and glycoproteomics have been developed and applied across laboratories depending on the glycoanalyte of interest (e.g., N-/O-/C-linked, glycan/glycopeptide/glycoprotein, labeled/unlabeled), the nature of the sample (e.g., cells, tissues, bodily fluids), amount (nanogram/microgram/milligram protein levels) and the analytical instrument available23,24,25,26,27,28,29,30,31,32. Despite the recognized benefits of their parallel use, glycomics and glycoproteomics are not commonly applied together but rather&#160;as stand-alone approaches in glycobiological and cancer research studies due to their high demand for analytical expertise and specialized instrumentation.
Recognizing this technology gap, more than a decade ago, we introduced the glycomics-assisted glycoproteomics method, which is capable of integrating glycomics and glycoproteomics data obtained from complex biological specimens33. In short, the glycomics-assisted (or glycomics-guided as in this protocol) glycoproteomics technology profiles the N- and/or O-glycans through an established porous graphitized carbon (PGC) LC-MS/MS method34,35, which are then used for the analysis of intact N- and/or O-glycopeptide data acquired from the same sample(s) by defining the boundaries of the glycan search space36. PGC-LC-MS/MS is a particularly powerful glycomics method as it provides quantitative insight into the glycome with high sensitivity and structural resolution, yielding information on topology (monosaccharide sequence and branching pattern) and glycosidic linkages of glycans even within complex mixtures35,37,38. The glycoproteomics workflow involves peptide generation, optional peptide labeling, multiplexing, and prefractionation, as well as enrichment before reversed-phase LC-MS/MS analysis that ideally employs different fragmentation methods including stepped collision energy/higher-energy collisional dissociation (sceHCD, for N-glycopeptides) and electron transfer/higher-energy collisional dissociation (EThcD, for O-glycopeptides) for analyte identification. For N-glycoproteome analysis, parallel de-N-glycoproteomics can guide the intact N-glycopeptide data analysis by defining the protein/peptide search space and by providing more details on the occupied N-glycosylation sites and their rate of site occupancy while the parallel analysis of non-modified peptides reveals any protein level changes between the studied conditions.
This paper provides a detailed and easy-to-follow step-by-step protocol for the glycomics-guided glycoproteomics method (see Figure 1 for workflow overview). The protocol provides experimental details of the sample preparation steps and the LC-MS/MS-based glycomics and glycoproteomics experiments and presents representative results demonstrating the application of the method to formalin-fixed paraffin-embedded (FFPE) tissue sections of tumors resected from colorectal cancer (CRC) patients, enabling insights into the glycobiology of the TME in a valuable sample type that is archived in cancer biobanks around the world. The analysis and integration of glycomics and glycoproteomics data are also briefly discussed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67405/67405fig01v2.jpg" /> 
Figure 1:&#160;Overview of the glycomics-guided glycoproteomics method. The overview shows detailed experimental steps in glycomics (left) and glycoproteomics (right) workflows, with an overview of the information obtained (bolded) and how the two approaches are integrated through improved data analysis and interpretation. Insert: Home-made SPE micro-columns used for the (i) glycomics and (ii-iii) glycoproteomics workflows. <a href="https://www.jove.com/files/ftp_upload/67405/67405fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ammonium acetate&amp;nbsp;</td><td>Sigma Aldrich</td><td>A1542</td><td>Alternatives available</td></tr><tr><td>Ammonium bicarbonate, purity &amp;ge;99.0%</td><td>Sigma Aldrich</td><td>A6141</td><td>Alternatives available</td></tr><tr><td>Anhydrous acetonitrile (ACN), LC-MS grade</td><td>Sigma Aldrich</td><td>34851</td><td>Alternatives available</td></tr><tr><td>Bovine fetuin</td><td>Sigma Aldrich</td><td>F3004</td><td>Alternatives available</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma Aldrich</td><td>D0632</td><td>Alternatives available</td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>E7023</td><td>Alternatives available</td></tr><tr><td>Formic acid, LC-MS grade</td><td>Sigma Aldrich</td><td>00940</td><td>Alternatives available</td></tr><tr><td>Glacial acetic acid</td><td>Sigma Aldrich</td><td>A6283</td><td>Alternatives available</td></tr><tr><td>Iodoacetamide (IAA)</td><td>Sigma Aldrich</td><td>I1149</td><td>Alternatives available</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>M1775</td><td>Alternatives available</td></tr><tr><td>Peptide-N-glycosidase F (PNGase F)</td><td>Promega</td><td>V4831</td><td>Elizabethkingia miricola PNGase F (10 U/&amp;mu;L) recombinantly expressed in Escherichia coli&amp;nbsp;</td></tr><tr><td>Polyvinylpyrrolidone 40 (PVP)</td><td>Sigma Aldrich</td><td>PVP40</td><td>Alternatives available</td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Sigma Aldrich</td><td>484016</td><td>Alternatives available</td></tr><tr><td>Sequencing-grade porcine trypsin&amp;nbsp;</td><td>Promega</td><td>V5113</td><td>Alternatives available</td></tr><tr><td>Sodium borohydride (NaBH&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>213462</td><td>Alternatives available</td></tr><tr><td>Triethylammonium bicarbonate (TEAB), LC-MS grade</td><td>Sigma Aldrich</td><td>T7408</td><td>Alternatives available</td></tr><tr><td>Trifluoroacetic acid (TFA), LC-MS grade</td><td>Sigma Aldrich</td><td>T6508</td><td>Alternatives available</td></tr><tr><td>&lt;strong&gt;Tools/Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C18 disks</td><td>Empore</td><td>66883-U</td><td></td></tr><tr><td>C8 disks</td><td>Empore</td><td>66882-U</td><td></td></tr><tr><td>Flat-bottom polypropylene 96-well plate&amp;nbsp;</td><td>Corning</td><td>3364</td><td></td></tr><tr><td>Immobilon-PSQ polyvinylidene difluoride (PVDF) membrane</td><td>Millipore</td><td>IPVH20200</td><td>Pore size: 0.45 &amp;mu;m&amp;nbsp;</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5452000069</td><td>Alternatives available</td></tr><tr><td>Microcentrifuge adapters&amp;nbsp;</td><td>The Nest Group, Inc., MA</td><td>SS18V</td><td>MiniSpin Column Collar, comes with Microspin Columns</td></tr><tr><td>Oligo R3 resin</td><td>Thermo</td><td>1133903</td><td>Particle size 30 &amp;mu;m</td></tr><tr><td>Parafilm</td><td>Parafilm M</td><td>PM996</td><td></td></tr><tr><td>Protein LoBind tubes 1.5 mL</td><td>Eppendorf</td><td>0030108450</td><td></td></tr><tr><td>Protein LoBind tubes 2.0 mL</td><td>Eppendorf</td><td>0030108442</td><td></td></tr><tr><td>Safe-lock tubes 1.5 mL</td><td>Eppendorf</td><td>0030120086</td><td></td></tr><tr><td>Safe-lock tubes 2.0 mL</td><td>Eppendorf</td><td>0030120094</td><td></td></tr><tr><td>Shaker</td><td>Eppendorf</td><td>5382000066</td><td>Alternatives available</td></tr><tr><td>Single hole puncher</td><td>Swingline</td><td>74005</td><td></td></tr><tr><td>SpeedVac concentrator</td><td>Martin Christ</td><td>RVC 2-25 CDplus</td><td>Alternatives available</td></tr><tr><td>Supelclean ENVI-Carb SPE resin</td><td>Supelco</td><td>57088</td><td>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</td></tr><tr><td>Syringe 5 mL</td><td>Sigma Aldrich</td><td>Z116866</td><td>Alternatives available</td></tr><tr><td>Temperature-controlled incubator</td><td>Thermoline</td><td>E7.30</td><td>Alternatives available</td></tr><tr><td>Ultrasonic bath</td><td>Unisonics</td><td>FXP</td><td>Alternatives available</td></tr><tr><td>ZIC-HILIC resin</td><td>Merck</td><td>150455</td><td>Particle/pore size, 5 &amp;mu;m/200 &amp;Aring;. Resin can be manually extracted from LC column, and can be stored long-term in 50% (v/v) methanol in MilliQ water</td></tr><tr><td>ZipTip C18 solid-phase extraction (SPE) micro-columns&amp;nbsp;</td><td>Millipore</td><td>ZTC18S096</td><td>Alternatively, home-made C18-SPE micro-columns can replace commercial product</td></tr><tr><td>&lt;strong&gt;LC-MS/MS Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1260 Infinity Capillary HPLC system&amp;nbsp;</td><td>Agilent</td><td></td><td>Alternatives available</td></tr><tr><td>Analytical LC column pre-packed with ReproSil-Pur C18 AQ resin</td><td>Dr Maisch, Ammerbuch-Entringen, Germany</td><td>r13.aq.s2570</td><td>Particle/pore size: 3 &amp;mu;m/120 &amp;Aring;, length/inner diameter: 25 cm/75 &amp;mu;m. Commercial C18 nano-LC columns also available/&amp;nbsp;</td></tr><tr><td>Dionex UltiMate 3000 RSLCnano LC System&amp;nbsp;</td><td>Thermo</td><td></td><td>Alternatives available</td></tr><tr><td>HyperCarb KAPPA PGC capillary LC column&amp;nbsp;</td><td>Thermo</td><td>Discontinued</td><td>Particle/pore size, 3 &amp;mu;m/250 &amp;Aring;; 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 &amp;mu;m/200 and with different column lengths and internal diameter are also available (e.g., Merck #59994-U).&amp;nbsp;</td></tr><tr><td>LCMS-grade acetonitrile</td><td>LiChrosolv</td><td>100029</td><td>Alternatives available</td></tr><tr><td>Linear trap quadrupole (LTQ) Velos Pro ion trap mass spectrometer (glycomics)&amp;nbsp;</td><td>Thermo</td><td></td><td>Alternatives available</td></tr><tr><td>Orbitrap Fusion Lumos Tribrid Mass Spectrometer (glycoproteomics)</td><td>Thermo</td><td></td><td>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)</td></tr><tr><td>Total Recovery Clear Glass screw vials 0.9 mL</td><td>Thermo</td><td>THC11093563</td><td>Alternatives available</td></tr><tr><td>Total Recovery Clear Glass screw vials matching caps</td><td>Thermo</td><td>THC09150869</td><td>Alternatives available</td></tr><tr><td>Trap LC column packed in-house with ReproSil-Pur C18 AQ resin&amp;nbsp;</td><td>Dr Maisch, Ammerbuch-Entringen, Germany</td><td>r15.aq.s0202</td><td>Particle/pore size: 5 &amp;mu;m/120 &amp;Aring;, length/inner diameter: 2 cm/100 &amp;mu;m. Commercial C18 trap LC columns also available.&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Byonic</td><td>Protein Metrics Inc</td><td>v2.6.46 or higher</td><td>Commercial glycopeptide and PTM search engine, accepting LC-MS/MS raw data from most MS vendors</td></tr><tr><td>Byos</td><td>Protein Metrics Inc</td><td>v3.9-7 or higher</td><td>Commercial software for manual inspection of glycopeptide candidates and the automatic annotation of glycan MS/MS spectra</td></tr><tr><td>GlycoMod</td><td>Expasy</td><td></td><td>Open access software, assisting the annotation of glycomics data (https://web.expasy.org/glycomod/)</td></tr><tr><td>GlycoWorkBench</td><td>EUROCarbDB</td><td>v2.1</td><td>Open access software, assisting the annotation of glycomics data and the drawing of glycan cartoons</td></tr><tr><td>RawMeat</td><td>VAST Scientific</td><td>v2.1</td><td>Open access software, extracting m/z of glycan precursor ions from raw spectral data</td></tr><tr><td>Skyline</td><td>Brendan X. MacLean</td><td>v21.2 or higher</td><td>Open access software for relative quantitation of glycans</td></tr><tr><td>Xcalibur</td><td>Thermo</td><td>v2.2 or higher</td><td>For browsing raw LC-MS/MS data</td></tr></tbody></table>

glycomics-guided glycoproteomics facilitates comprehensive profiling of the glycoproteome in complex tumor microenvironments

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

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.

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)<sup c...

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-guided-glycoproteomics-facilitates-comprehensive-profiling

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Learn glycomics-guided glycoproteomics for comprehensive tumor microenvironment profiling using mass spectrometry. This method enables precise glycopeptide analysis and reveals cancer glycobiology insights.

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

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