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

Summary

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.

Abstract

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.

Introduction

Protein glycosylation is a prevalent and complex type of co- and post-translational modification of proteins produced by species across the phylogenetic tree of life^1,2,3. 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 events^4,5. Aberrant protein glycosylation has been thought to be a cause of malignant transformation, tumor progression, and spread^6,....

Protocol

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....

Discussion

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 .......

Materials

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