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

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...

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

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

Research

JoVE Journal

Cancer Research

971 Views.  Macquarie University. 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.

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

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Biochemistry

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

Chemistry

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 &#181;m and 300 &#181;m diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 &#181;m.

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically complex tumors, with a high degree of proteomic variability across both the tumor itself and its heterogenous microenvironment. The malignant potential of these tumors is enhanced by the dysregulation of proteins involved in several key pathways, including processes that maintain cellular stability and preserve the structural integrity of the microenvironment. Although there have been numerous bulk and single-cell glioma analyses, there is a relative paucity of spatial stratification of these proteomic data. Understanding differences in spatial distribution of tumorigenic factors and immune cell populations between the intrinsic tumor, invasive edge, and microenvironment offers valuable insight into the mechanisms underlying tumor proliferation and propagation. Digital spatial profiling (DSP) represents a powerful technology that can form the foundation for these important multilayer analyses.
DSP is a method that efficiently quantifies protein expression within user-specified spatial regions in a tissue specimen. DSP is ideal for studying the differential expression of multiple proteins within and across regions of distinction, enabling multiple levels of quantitative and qualitative analysis. The DSP protocol is systematic and user-friendly, allowing for customized spatial analysis of proteomic data. In this experiment, tissue microarrays are constructed from archived glioblastoma core biopsies. Next, a panel of antibodies is selected, targeting proteins of interest within the sample. The antibodies, which are preconjugated to UV-photocleavable DNA oligonucleotides, are then incubated with the tissue sample overnight. Under fluorescence microscopy visualization of the antibodies, regions of interest (ROIs) within which to quantify protein expression are defined with the samples. UV light is then directed at each ROI, cleaving the DNA oligonucleotides. The oligonucleotides are microaspirated and counted within each ROI, quantifying the corresponding protein on a spatial basis.

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Medicine

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Biology

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...