This work was supported by grants from Institut National du Cancer, FRANCE, INCa grant PLBIO16-251 (PLBIO16-251), CONACyT-SRE (Mexico) grant 0280365 and the REPERE program of Occitanie, France (M.S.R.). Also, NIH (US Department of Health and Human services)-R01AR001576-11A1, Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco, MINECO: SAF2017-87301-R integrado en el Plan Estatal de Investigaci&#243;n Cientifica y T&#233;cnica y Innovaci&#243;n 2013-2016 cofinanciado con Fondos FEDER, BIOEF (Basque Foundation for Innovation and Health Research): EITB Maratoia BIO15/CA/014; Instituto de Salud Carlos III:PIE14/00031, integrado en el Plan Estatal de Investigaci&#243;n Cientifica y T&#233;cnica y Innovaci&#243;n 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C), Asociaci&#243;n Espa&#241;ola contra el C&#225;ncer (T.C.D, M.L.M-C), Daniel Alagille award from EASL (to T.C.D),&#160;Fundaci&#243;n Cient&#237;fica de la Asociaci&#243;n Espa&#241;ola Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M), La Caixa Foundation Program (to M.L.M). We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644).

All experiments were approved by the CIC bioGUNE institutional committees for animal care and handling. All efforts were made to minimize animal suffering and to reduce the number of animals used. 3-months old male glycine N-methyltransferase (Gnmt) deficient (Gnmt&#8722;/&#8722;) and its wild type littermates (Gnmt+/+) were used.
1. Cell Preparation and Lysis
NOTE: Herein, Huh-7 (human hepatoma cell line) and THLE2 (human hepatic cell line) were used.
<ol>
	<li>Maintain cells in P100 plates in standard growth media at 37&#8239;&#176;C in a humidified atmosphere of 5% CO2-95% humidity.</li>
	<li>Grow cells in P100 plates plating at a density of 1.2&#8211;1.5 &#215; 106 cells per dish by counting the cells using a Neubauer haemocytometry counting chamber.
	<ol>
		<li>Culture Huh-7 in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin-amphotericin B (PSA) and 1% glutamine.</li>
		<li>Culture THLE2 cells in culture dishes pre-coated with 0.01 mg/mL fibronectin, 0.01 mg/mL bovine serum albumin (BSA) and 0.03 mg/mL collagen type I dissolved in growth medium which consists of bronchial epithelial cell growth basal medium (BEGM) supplemented with growth factors (0.4% BPE, 0.1% insulin, 0.1% hydrocortisone, 0.1% retinoic acid, 0.1% transferrin, 0.1% triiodothyronine as well as 10% FBS, 1% PSA, 5 ng/mL epidermal growth factor (EGF) and 70 ng/mL phosphoethanolamine.</li>
	</ol>
	</li>
	<li>At the end point of the experiment, aspirate the media from the plates and wash cells with 5 mL of sterile 1x phosphate-buffered saline (PBS). Lyse cells directly on the plate placed on ice using 500 &#181;L of lysis buffer (50 mM Tris pH 8.5, 150 mM NaCl, 5 mM EDTA, 1% nonidet P-40 (NP40), supplemented with complete EDTA-free protease inhibitor cocktail and 50 &#956;M PR-619 for each P100 dish. Using a cell scraper, gently scrape the cells off the bottom of the plate into the Lysis medium. 
	NOTE: Check that all the cells have detached from the plate by visually inspecting the plate base after the treatment.</li>
	<li>Alternatively, harvest cells by trypsinization by aspirating cell media and add 1 mL of 1x (0.05%) trypsin-EDTA to the plate, enough to cover the cells and placing the plate in the incubator set at 37 &#176;C, 5% CO2, and 95% humidity for ~5 min ensuring all cells have detached from the plate. Add 2 mL of pre-warmed growth medium in order to stop trypsinization. Centrifuge at 150 g for 10 min and aspirate the supernatant. Wash with 1x PBS and centrifuge at 150 x g for 10 min. After aspirating the supernatant, add 500 &#181;L of lysis buffer (50 mM Tris pH 8.5, 150 mM NaCl, 5 mM EDTA, 1% NP40, supplemented with complete EDTA-free protease inhibitor cocktail and 50 &#956;M PR-619 for each P100 dish. 
	NOTE: The addition of the PR-619 is critical.</li>
	<li>Centrifuge at 15,500 x g and 4&#176;C for 10 min. Transfer the supernatant to another tube and discard the pellet. 
	NOTE: The protocol can be paused here, and samples stored at -80 &#176;C until further analysis.</li>
</ol>
2. Tissue Preparation and Lysis
<ol>
	<li>Upon animal sacrifice, collect mouse livers, wash with cold PBS, and snap freeze immediately in liquid nitrogen. Store the samples &#8722;80 &#176;C until further analysis.</li>
	<li>Homogenize 75 mg fragments from snap-frozen/or fresh livers in 1 mL of ice-cold lysis buffer (50 mM Tris pH 8.5, 150 mM NaCl, 5 mM EDTA, 1% NP40, supplemented with complete EDTA-free protease inhibitor cocktail and 50 &#956;M PR-619). Run the homogenizer at 6500 x rpm, 2 x 60 s each, with a 30 s pause (see Table of Materials).</li>
	<li>Centrifuge the samples in a microfuge at 15, 500 x g and 4 &#176;C for 10 min. Transfer the supernatant to another tube and discard the pellet.</li>
	<li>Alternatively, triturate 75 mg of frozen tissues in liquid nitrogen. Then recover the tissue in 1 mL of lysis buffer.</li>
	<li>Centrifuge the sample in a microfuge at 15, 500 x g and 4 &#176;C for 10 min. Transfer the supernatant to another tube and discard the pellet. 
	NOTE: The protocol can be paused here, and samples stored at -80 &#176;C until further analysis.</li>
</ol>
3. Binding of GST-SUBEs or GST Control to Glutathione-Agarose Beads
NOTE: The synthesis of the GST-SUBEs or GST control are outside the scope of this manuscript and can be reviewed in previously published literature20. Alternatively, GST- and Control SUBEs are commercially available (e.g., SignalChem).
<ol>
	<li>Preparation of glutathione beads
	<ol>
		<li>Add 1 mL of de-ionized water to 70 mg of lyophilized glutathione-agarose beads. Reconstitute the beads overnight at 4 &#176;C (or for at least 30 min at room temperature).</li>
		<li>Wash the beads thoroughly after swelling (to remove lactose and ethanol that are usually present in the lyophilized powder agarose beads). To do so, first wash with 10 mL of de-ionized water or PBS followed by centrifugation at 300 x g for 5 min at room temperature. Perform this thrice.</li>
		<li>After 3 washes, resuspend the beads in 1 mL of PBS to obtain a 50% (v/v) slurry. 
		NOTE: This volume is suitable for the analysis of 10 samples.</li>
	</ol>
	</li>
	<li>For each sample, add 100 &#956;g of GST-SUBEs or GST control (see reference20) to 100 &#956;L of the glutathione beads slurry and 500 &#181;L of PBS. 
	NOTE: The relative abundance of the SUMOylated proteins of interest determines the amount of GST-SUBEs used for pull-downs. For each new experimental model, analyze the condition prior to the actual experiment by western blotting the input, bound, and flow-through (FT) material using anti-SUMO2/3 antibodies or against proteins of interest (Liver Kinase B1 (LKB1).</li>
	<li>Incubate all the GST-SUBEs or GST control with beads prepared in 3.2., slowly rotating in rotator or mini roller (see Table of Materials) at 4 &#176;C for at least 2 h (slow binding reaction). 
	NOTE: Adding 1 mM dithiothreitol (DTT) improves GST binding to the glutathione beads.</li>
	<li>Recover the agarose beads by centrifugation at 300 x g for 5 min at room temperature. At the end, resuspended the beads in PBS to obtain 50% (v/v) slurry. 
	NOTE: The protocol can be paused here, and samples stored at -80 &#730;C until further analysis.</li>
</ol>
4. GST Pull Down Assay
<ol>
	<li>After step 1.5, 2.3 or 2.5, take 1/10 of total volume (e.g. 50 &#956;L) and dilute in the same volume of 3x boiling buffer (250 mM Tris-HCl pH 6.8, 500 mM &#946;-mercaptoethanol, 50% glycerol, 10% SDS, bromophenol blue). This fraction is considered as INPUT.</li>
	<li>Add 450 &#956;L of clarified lysate from steps 1.5, 2.3 or 2.5 to 100 &#956;L glutathione beads slurry. Incubate the lysate with beads, slowly rotating at 4 &#176;C for at least 2 h. 
	NOTE: Alternatively, 100-200 &#956;g of total protein from steps 1.5, 2.3 or 2.5 (quantified with the Bradford assay) in a total volume of 450 &#956;L can be used.</li>
	<li>Spin down the beads in a microfuge at 300 x g for 5 min and collect the supernatant for the analysis. Transfer 1/10 of the total volume (e.g., 50 &#956;L) in a separate tube and dilute in an equal volume of 3x boiling buffer. This fraction is the flow-through (FT) fraction.</li>
	<li>Wash the remaining sample three times with 1 mL ice-cold PBS, 0.05% Tween 20, spin down at 4 &#176;C and 300 x g for 1 min. Carefully aspirate ensuring no liquid remains. The beads correspond to SUBEs BOUND (SB) fraction.</li>
	<li>Elute the sample with 15 &#956;L of 3x boiling buffer and 15 &#956;L of the lysis buffer. This is called the BOUND Fraction.</li>
</ol>
5. Identification and Characterization of SUMO Targets by Western Blot analysis
<ol>
	<li>Perform western blot analysis using an anti-SUMO2/3 antibody or any other specific antibody of choice as described previously22.</li>
</ol>
6. Identification and Characterization of the SUMOylated Proteome by Mass Spectrometry
NOTE: In the case of Mass-Spectrometry (MS) analysis, samples were processed using the Filter-Aided Sample Preparation (FASP) method described by Wisniewski et al.23.
<ol>
	<li>Desalt the peptides by using stage-tip C18 microcolumns and resuspend them in 0.1% formic acid (FA) prior to MS analysis.</li>
	<li>Load the samples onto a LC-MS system (see Table of Materials) and analyze them in triplicate (technical replicates) (Figure 2b).</li>
	<li>Carry on with the protein identification and abundance calculation by using an associated software.</li>
	<li>For statistical analysis and heatmap generation, load the data onto the Perseus platform (http://www.perseus.tufts.edu/hopper/). Apply a permutation-based false discovery rate (FDR)-corrected t-test for the comparison of the abundances. Proteins with a q &#60; 0.05 and a SUBEs/GST ratio greater than 2 were considered as enriched24. 
	NOTE: Proteins identified with at least two different peptides are considered in the final analysis.</li>
</ol>

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Dr. Mart&#237;nez-Chantar advises for Mitotherapeutix LLC.

Herein, we have provided a complete and detailed description of the methodology reporting the use of SUBEs for the enrichment, isolation and identification and characterization of the SUMOylated proteins in in vivo models of liver cancer. Both in mouse liver tumors and human hepatoma cells, we were able to correctly isolate and identify SUMOylated proteins of interest and to perform a high-throughput characterization of the SUMOylated proteome and interactome. Even though the synthesis of SUBEs is outside the scope of this manuscript, for further information the following references should be looked at26. The protocol described is fast and very sensitive and the critical step of the protocol includes the use of SENPs inhibitors (PR-619). In alternative, chemical isopeptidase inhibitors such as NEM (N-Ethylmaleimide) and IAA (2-Iodoacetamide) in the lysis buffer can be used, however, previous reports have shown that for the SUBEs protocol, the use of PR-619 is advantageous as the other inhibitors interfere with GST binding to the glutathione beads20.
SUBEs are recombinant proteins that comprise tandem repeats of SIMs thereby recognizing SUMO molecules on modified proteins with an increase in the overall affinity for SUMO substrates. Due to its high specificity and sensitivity, the use of SUBEs for the isolation of the SUMOylated proteome is advantageous relative to other approaches in the literature such as the detection by western-blot of specific SUMOylated proteins using antibodies against the protein of interest or the nickel chromatography using the different histidine-tagged versions of SUMO molecules. However, it must be taken into consideration that as the SUBEs protocol is performed under non-denaturing conditions, the interaction between SUMOylated proteins and other interacting proteins is maintained. Therefore, we obtain information about the SUMO interactome rather than only a list of SUMOylated target proteins. Thus, further experiments are necessary to confirm if the identified protein is a SUMO target or an interacting factor. Other limitation of the SUBEs is the fact that control GST traps used are able to capture many background proteins related to oxidative stress. This issue is especially relevant during the MS analysis due to the high sensitivity of the technique. In order to overcome these limitations, biotinylated SUMO-traps (bioSUBEs) have been developed26. Another limitation of SUBEs resides in the fact that we are only able to capture proteins modified by SUMO 2 and SUMO 3 whereas SUMO 1-modified proteins cannot be isolated.
Other concern of the use of SUBEs is related to the amount of starting material necessary for the procedure. The starting material used to capture SUMOylated proteins should consider the different experimental conditions explored. While basal SUMOylation has been reported in various cellular contexts, SUMOylation is a process that is strongly induced after multiple stress conditions/stimuli. If comparing untreated versus treated samples, one has to be sure that the column is not saturated, and differences can be observed between those conditions. In the case of the mouse phenotypes we are analyzing, no treatments have been used and basal SUMOylation levels are low. For this reason, high amounts of proteins were used. The background level should be controlled by using GST and if the unspecific binding is high, the amount of starting material or the binding time should be reduced. The analysis of the FT fraction can be indicative of the capture efficiency even if these traps prefer poly-SUMOylated proteins and a total depletion should not be expected, a reduction of total SUMOylation is in general well observed when the capture efficiency is optimal.&#160;
Finally, other application of the SUBEs technology includes the combination of SUBEs technology with Real-time Surface Plasmon Resonance (SPR) allowing the real-time interactions with SUMOylated proteins from cell extracts27. Also, more recently, biotinylated SUMO-traps (bioSUBEs) have been developed with the advantage to reduce the background associated to bigger tags, e.g., during mass spectrometry analysis26. In addition, the bioSUBE version can be used to detect SUMOylated proteins in living cells by fluorescence by using streptavidin-labelled with distinct fluorescent dyes taking advantage of the streptavidin binding to biotin. Also, methods for detection and quantification of SUMOylated proteins can be considered with both GST and bioSUBEs versions such as it was done with the tandem ubiquitin binding entities (TUBEs)21.
Overall, the use of SUBEs for the isolation and characterization of the SUMOylated proteome relevant in liver cancer is a fast and sensitive method providing vast information on the still rather unknown role of the SUMOylation pathway in liver cancer.

Liver cancer is the sixth most common cancer worldwide and the second cause of cancer-associated deaths1. Hepatocellular Carcinoma (HCC) is the most prevailing form of primary liver cancer. Historically, common risk factors for the development of HCC included chronic hepatitis B or C infection and abusive alcohol consumption. In the last decades, the metabolic syndrome, Type&#160;2 diabetes non-alcoholic fatty liver disease (NAFLD) has emerged as risk factors for the development of HCC2. HCC is very heterogeneous, both phenotypically and genetically, wherein a complex network of signaling pathways are disrupted. In the last years, even though there has been an increase in our knowledge about the molecular pathways implicated in the pathogenesis of HCC, there are still no effective therapeutic approaches for HCC management. Many pathways are activated in HCC and inhibiting one generally drives the compensation by other pathways3. This has been one of the main difficulties when treating HCC. Thus, a more global approach may provide a potential therapeutic approach for the clinical management of liver cancer e.g., targeting post-translational modifications (PTMs), as multiple signaling pathways can be simultaneously regulated by PTMs of proteins.
Post-translational modifications are considered as key mechanisms regulating protein homeostasis and functions4. Structural and functional changes are introduced by PTMs, thereby, increasing proteome diversity. The most common PTMs include phosphorylation, methylation, acetylation, glycosylation, ubiquitination, and conjugation of ubiquitin-like proteins (UbLs). Among all UbLs, protein modification by SUMO (Small Ubiquitin MOdifier) has attracted attention in association with its critical role in a variety of cellular processes, including transcription, cellular localization, DNA repair, and cell cycle progression5. Recently, SUMOylation was shown to be altered in liver cancer6,7,8,9, and changes in the SUMOylation of specific proteins has been described to play a role in the progression of cancer-related diseases9.
In mammals, there are five SUMO paralogues, SUMO-1 to SUMO-5. To date, no experimental evidence is available about the existence of endogenous SUMO-4 and endogenous SUMO-5 conjugation reactions at the protein level10,11,12. SUMOylation in mammals is carried out by an enzymatic thiol-ester cascade involving three enzymes, the heterodimeric SUMO activating enzyme (SAE1/SAE2) or E1, the SUMO conjugating enzyme (Ubc9) or E2 and a SUMO-E3-ligase specific for each target protein. The action of several families of SUMO E3s appears to be in a dynamic equilibrium with SUMO-specific proteases (SUSPs or SENPs)13 making the SUMOylation reaction highly reversible. Moreover, only a small fraction of the SUMOylated protein versus non-SUMOylated total protein is present. Thereby, isolating endogenous SUMOylated proteins in vivo is rather challenging13.
SUMOylation in vivo was initially studied by western blot using antibodies against the protein of interest14. Immunoprecipitation of the protein was performed with specific antibodies and then PAGE-western blot was carried out with anti-SUMO antibodies. The main problem with this strategy is that antibodies generated against a non-modified recombinant protein are not always able to immunoprecipitate the SUMOylated form of a protein. Alternatively, nickel chromatography after the transient expression of histidine tagged (His6) versions of SUMO molecules and the protein of interest has been used to study SUMOylation in cells. On this basis, it will be more convenient to detect SUMO-modified forms from cells stably expressing His6-SUMO15. For in vivo studies, tandem-SUMO-interacting motifs (SIM) based enrichment was demonstrated for the purification of polySUMO conjugates16. Other groups have been using epitope-tagged antibody SUMO approaches providing a feasible tool to investigate endogenous SUMOylation in primary cells, tissues, and organs17,18. And more recently, Nielsen and colleagues have used antibody-based enrichment to identify endogenous and site-specific SUMO in cells and tissues19.
In order to provide complementary information on the role of SUMOylation in vivo, SUMO-binding entities (SUBEs), also known as SUMO traps, were developed20. Of relevance, tandem ubiquitin binding entities (TUBEs) are considered the conceptual precursors of SUBEs and are commercially available tools for the detection and isolation of polyubiquitylated proteins21. SUBEs are recombinant proteins that comprise tandem repeats of SIMs thereby recognizing SUMO molecules on modified proteins with an increase in the overall affinity for SUMO substrates. SUMO-traps were engineered by introducing an E3 ubiquitin-protein ligase RNF4-derived SIM2 and SIM3 motifs in tandem, into a vector containing glutathione S-transferase (GST), a heterologous carrier protein20. Although SUBEs cannot be used properly to identify mono-SUMOylated target proteins, this method provides a tool to facilitate the purification and identification of poly-SUMO target proteins in vivo. Herein, we describe the application of SUBEs to isolate SUMOylated proteins both in human hepatoma cells and in mouse liver biopsies, an important tool for the study of liver cancer. An overall scheme of the protocol described in this manuscript is shown in Figure 1.

<table>
	<tbody>
		<tr>
			<td>(Gnmt&#8722;/&#8722;)/ (Gnmt+/+) mice</td>
			<td>CIC bioGUNE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>BEBM</td>
			<td>Lonza/Clonetics Corporation</td>
			<td>cc-3171</td>
			<td></td>
		</tr>
		<tr>
			<td>BEGM Bullet Kit</td>
			<td>Lonza/Clonetics Corporation</td>
			<td>CC3170</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma</td>
			<td>115-39-9</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 microcolumns</td>
			<td>Millipore</td>
			<td>Z720070</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen type I</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-136157</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete tablets EDTA-free</td>
			<td>Roche</td>
			<td>4693132001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>A14431-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>43815</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma</td>
			<td>e9644</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Life Technologies</td>
			<td>33010018</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione agarose beads</td>
			<td>Sigma</td>
			<td>G4510</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>GST-Control</td>
			<td>SignalChem</td>
			<td>G52-30H</td>
			<td></td>
		</tr>
		<tr>
			<td>GST-SUBEs</td>
			<td>SignalChem</td>
			<td>S291-340G</td>
			<td></td>
		</tr>
		<tr>
			<td>Huh7</td>
			<td>CLS (Cell Lines Service)</td>
			<td>300156</td>
			<td>https://clsgmbh.de/</td>
		</tr>
		<tr>
			<td>IAA (2-Iodoacetamide)</td>
			<td>Merck</td>
			<td>L58046844</td>
			<td></td>
		</tr>
		<tr>
			<td>LKB1 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32245</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini LabRoller Rotator</td>
			<td>LABNET</td>
			<td>H5500</td>
			<td>https://www.labnetinternational.com</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Merck</td>
			<td>106404041000</td>
			<td></td>
		</tr>
		<tr>
			<td>nanoElute</td>
			<td>BRUKER</td>
			<td></td>
			<td>https://www.bruker.com/</td>
		</tr>
		<tr>
			<td>NEM (N-Ethylmaleimide)</td>
			<td>Sigma</td>
			<td>E3876</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40</td>
			<td>Fluka</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Peaks software</td>
			<td>Bioinformatics Solutions Inc.</td>
			<td></td>
			<td>http://www.bioinfor.com/</td>
		</tr>
		<tr>
			<td>Phosphoetanolamine</td>
			<td>Sigma</td>
			<td>P0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S solution</td>
			<td>Sigma</td>
			<td>P7170</td>
			<td></td>
		</tr>
		<tr>
			<td>PR-619</td>
			<td>Merck</td>
			<td>662141</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys 24</td>
			<td>Bertin Technologies</td>
			<td>P000669-PR240-A</td>
			<td></td>
		</tr>
		<tr>
			<td>PSA</td>
			<td>Life Technologies</td>
			<td>151-40-122</td>
			<td></td>
		</tr>
		<tr>
			<td>PSG</td>
			<td>Life Technologies</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>SUMO2/3 antibody</td>
			<td>Abcam</td>
			<td>Ab3742</td>
			<td></td>
		</tr>
		<tr>
			<td>THLE-2</td>
			<td>ATCC</td>
			<td>ATCC CRL-2706</td>
			<td>http://www.lgcstandards-atcc.org</td>
		</tr>
		<tr>
			<td>timsTOF Pro with PASEF mass spectrometer</td>
			<td>BRUKER</td>
			<td></td>
			<td>https://www.bruker.com/</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma</td>
			<td>60-24-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

sumo-binding entities (subes) as tools for the enrichment, isolation, identification, and characterization of the sumo proteome in liver cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

Identification of a Specific SUMOylation Substrate in Liver Tumor Biopsies by Western Blot Analysis
Liver Kinase B1 (LKB1) SUMOylation has been recently shown to be an important oncogenic driver in liver cancer9,25. Mice deficient in glycine N-methyltransferase (Gnmt), often referred to as Gnmt&#8722;/&#8722;, is a model that spontaneously develops hepatocellular carcinoma (HCC), the most common type of primary liver cancer. SUBEs were used to enrich and isolate the SUMOylated proteins both in Gnmt&#8722;/&#8722; mice with liver cancer mice and its wild type littermates (Gnmt+/+). In Figure 2a, Ponceau S staining of the three different fractions (input, FT and BOUND) obtained in the SUBEs pull-down assay are included. A Ponceau&#160;S stain is useful to control a possible deleterious effect on the loading of blotted proteins to be evaluated by Western blots. In Figure 2b a Western blot analysis of LKB1 by using SUBEs to capture endogenous SUMOylated LKB1 is shown. The levels of LKB1 SUMOylation are augmented in liver tumors. In the case of Western blot analysis, equal loads and transferred proteins were observed by Ponceau staining of the input fraction and were not significantly altered after washes (flow through fraction). The amount of protein captured with SUBEs was significantly higher, particularly in the tumors. Alternatively, staining a duplicate gel with Coomassie blue can provide similar information. Sticky proteins such as p53 or SUMOylated forms of some proteins might bind to the GST control. To remove the background, use low-density agarose beads, perform a coating with BSA, or incorporate additional washes. However, this could affect applications such as Mass Spectrometry and might result in loss of low affinity interacting proteins.
Characterization of the SUMO Interactome in Human Hepatoma Cells by Mass Spectrometry Analysis
To investigate the capacity of the SUMO-trap to interact with naturally SUMOylated proteins, Huh-7 (human hepatoma) and non-transformed liver epithelial human THLE2 cell lines were used. The first step is the visualization of the total material captured with SUBEs and using GST as a negative control. For this purpose, we can use conventional protein staining protocols as shown in Figure 3a. Then, we performed mass spectrometry analysis. An average of 2268 proteins were identified in the Huh7 GST samples (2339, 2297 and 2168 for each load, respectively), whereas 2812 proteins were identified on average in the Huh7 SUBEs sample (2815, 2817 and 2806). After the subtraction, 742 proteins were enriched in the SUBEs. On the other hand, an average of 2497 proteins were identified in the THLE2 GST samples (2476, 2520 and 2495, respectively) and 2763 in the SUBEs (2823, 2783 and 2684). Of these, 577 were considered to be enriched in the SUBEs samples. Analysis of technical replicates retrieves the heatmap shown in Figure 3b, which was calculated using the default settings available (Euclidean distance, average linkage and pre-processed with k-means). The heatmap depicts the distribution of the 100 most significantly and exclusively enriched proteins in each cell line.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60098/60098fig01.jpg" /> 
Figure 1: Schematic diagram of the protocol flow chart used for the enrichment, isolation and identification and characterization of the SUMOylated proteome in vivo for the study of liver cancer. <a href="https://www.jove.com/files/ftp_upload/60098/60098fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60098/60098fig02.jpg" /> 
Figure 2: Modification of LKB1 by SUMO-2 in mouse models of Hepatocellular Carcinoma. 
(a) Ponceau S staining of the three different fractions (input, Flow through (FT) and BOUND) obtained in the SUBEs pull-down assay. (b) Western blot analysis of LKB1 by using SUMO binding entities (SUBEs) to capture endogenous SUMOylated LKB1; GAPDH is used as a loading control. <a href="https://www.jove.com/files/ftp_upload/60098/60098fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60098/60098fig03.jpg" /> 
Figure 3: Differences between the SUMOylated proteome between the tumoral Huh-7 and non-transformed liver epithelial THLE2 human cell lines. 
(a) Sypro staining of captured protein material, with GST (Negative control) and SUBEs. (c) Heatmap depicting the differentially enriched proteins in Huh-7 and THLE2 SUBE samples. <a href="https://www.jove.com/files/ftp_upload/60098/60098fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer at JoVE.com

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

sumo-binding-entities-subes-as-tools-for-enrichment-isolation

Research

JoVE Journal

Biochemistry

7.0K Views.  CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd). Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Video: SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results.
Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY&#160; II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input.
Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Crystallographic fragment screening at the Helmholtz-Zentrum Berlin is performed using a workflow with dedicated compound libraries, crystal handling tools, fast data collection facilities&#160;and largely automated data analysis. The presented protocol intends to maximize the output of such experiments to provide promising starting points for downstream structure-based ligand design.

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...