This work was supported by the grant P01HL107151 from the NHLBI/NIH. The authors also gratefully acknowledge the support and access to instrumentation provided through grant P41GM103490 from the NIGMS/NIH.

NOTE: Lab Safety Concerns
In keeping with standard laboratory best practices, observe the following. Store all organic solvents in appropriate locations. Keep all waste materials in chemical waste containers with clear labeling of chemical compositions. As several reagents used in these protocols are potential carcinogens or generate volatile combustible gases, handle all reagents in a fume hood with ventilation. Wear personal protective equipment such as gloves, lab coat, and eye protection when working with organic solvents.
<img alt="Figure 1" src="/files/ftp_upload/51840/51840fig1highres.jpg" width="450px" /> 
Figure 1. Procedure for in-gel O-glycomics. (A) Proteins of interest are resolved by SDS-PAGE, detected by appropriate staining procedures (Coomassie or silver), and excised. Excised gel pieces are sliced into small cubes, destained, and washed with ethyl acetate to reduce contaminants that interfere with subsequent MS analysis. A portion of the gel slice can be reserved for in-gel tryptic digestion and subsequent proteomic characterization by LC-MS/MS. (B) O-linked glycans are released from resolved glycoproteins by in-gel reductive &#946;-elimination. Essential steps include desalting and borate removal by azeotrope with methanol. (C) O-linked glycans released by reductive &#946;-elimination are permethylated and subsequently partitioned into aqueous and organic phases. Sulfoglycans are quantitatively recovered in the upper (aqueous) phase while neutral and sialylated glycans partition into the lower (DCM) layer.
<img alt="Figure 2" src="/files/ftp_upload/51840/51840fig2highres.jpg" width="700px" /> 
Figure 2. Detail flow chart for in-gel glycomics. Each experimental step shown in Figure 1 is individually illustrated. (A) gel destaining and EtOAc extraction, (B) direct in-gel reductive &#946;-elimination for O-linked glycan release, and (C) permethylation and phase partition. <a href="/files/ftp_upload/51840/51840fig2highres.jpg" target="_blank">Please click here to view a larger version of the figure.</a>

1. Gel Excision and Removal of Gel-derived Contaminants
<ol>
	<li>Before starting the procedure, prepare chemicals and reagents listed in Materials List.</li>
	<li>Resolve protein on SDS-PAGE gel using standard Tris-Glycine buffer systems. Stain resolved proteins with either Coomassie Brilliant Blue or Silver Stain. In general, recovery of O-linked glycans from silver stained gels is approximately 80&#8211;90% of the recovery achieved from Coomassie stained gels.</li>
	<li>After electrophoresis, place the gel on a glass plate and excise the region of interest using a clean scalpel or razor blade.</li>
	<li>To increase yield of O-glycans, transfer the gel band of interest onto another glass plate and cut the excised gel piece into approximately 2 mm cubes. Avoid making gel pieces smaller than 1 mm cubes because glycan recovery is reduced. Transfer the small gel pieces into a screw top glass tube (13 x 100 mm) with a stainless steel microspatula.</li>
	<li>Add 1 ml of 25 mM ammonium bicarbonate (AMBIC, NH4HCO3) to the sample tube, cap the glass tube with a Teflon-lined screw top cap, and gently mix by flicking the tube before letting stand for 10 min. Do not employ vigorous agitation in order to avoid ripping the gel pieces.</li>
	<li>Carefully remove AMBIC from the glass tube using either a Pasteur glass pipette or a pipette equipped with an extra-long plastic tip. Avoid smashing and transferring the small gel pieces while removing the AMBIC solution.</li>
	<li>Add 1 ml of acetonitrile (ACN) to the glass tube, cap, and gently mix by flicking tube before letting stand for 10 min. Ensure that the gel pieces are completely covered with solvent. If necessary, push gel pieces off of tube wall and into solvent with pipette tip.</li>
	<li>Pipette off ACN from the glass tube and repeat steps 1.5&#8211;1.7 until the bright blue color is eliminated. Repeat a minimum of five sequential AMBIC/ACN washes if necessary. For silver stained gels, use a destain kit. Proceed to the next step when the gel piece turns uniformly white while in ACN.</li>
	<li>Pipette off any liquid from the glass tube, recap the tube, add 1 ml of AMBIC and let stand for 5 min.</li>
	<li>Remove AMBIC and add 2 ml of ethyl acetate (EtOAc) to the glass tube. Recap tube with Teflon-lined cap and place at 4 &#176;C overnight with end-over-end agitation or alternatively perform three EtOAc washes at room temperature for 30 min each. The longer the EtOAc wash, the more efficient the removal of contaminants.</li>
	<li>Remove the final EtOAc wash and perform three washes each with 2 ml of H2O. Complete removal of EtOAc will ensure a robust reductive &#946;-elimination reaction.</li>
	<li>Pipette off H2O and dehydrate the gel by washing once with 1 ml of ACN. 
	NOTE: After drying, the dehydrated gels are ready for in-gel &#946;-elimination. Dehydrated gel pieces can be stored at -20 &#176;C until use.</li>
	<li>Stop the washing process at any step and store the gel pieces at 4 &#176;C overnight, regardless of which buffer or solution is applied to the gel (AMBIC, ACN, H2O). Restart sample preparation anytime within the next 2 days.</li>
	<li>Use a portion of the destained and dehydrated gel pieces for protein ID by standard proteomic approaches.</li>
</ol>
2. In-gel O-glycan Release by Reductive &#946;-Elimination
<ol>
	<li>Prepare a stock solution of 1 M sodium hydroxide (NaOH) using 50% NaOH or solid NaOH and store at 4 &#176;C until use.
	<ol>
		<li>Take 5.2 ml of 50% NaOH (or 4 g of solid NaOH) and adjust volume to 100 ml to make 1 M NaOH solution. Dilute the 1 M NaOH stock solution to 100 mM NaOH, which can be stored at 4 &#176;C up to 6 months without loss of efficiency in reductive &#946;-elimination reactions.</li>
	</ol>
	</li>
	<li>Make 2 M sodium borohydride (NaBH4) in 100 mM NaOH. Dissolve 38 mg of NaBH4 in 0.5 ml of 100 mM NaOH. Generally, use 0.5&#8211;1.0 ml of the borohydride solution for each sample. Vary the volume of the reaction solution on the amount of dried gel pieces prepared in step 1. Expect to use 0.5 ml of borohydride solution for 1&#8211;2 excised gel pieces (step 1.3).</li>
	<li>Add 500 &#181;l of 100 mM NaOH to the dehydrated gel pieces and let stand for 3&#8211;5 min on ice in order to equilibrate the gel to the basic conditions which enhance glycan recovery.</li>
	<li>Add 500 &#181;l of 2 M NaBH4 in 100 mM NaOH, resulting in final concentrations of 1 M NaBH4 in 100 mM NaOH. Gently mix the tube and incubate at 45 &#176;C for 18 hr.
	<ol>
		<li>During the first hour of incubation, gently mix the tube every 15 min. Avoid strong agitation, because it may fragment the gel pieces. Perform the reaction in a well-equilibrated incubator/oven or in a heating block. Ensure that the gel pieces are covered with reaction solution.</li>
	</ol>
	</li>
	<li>Stop the reaction by removing the sample tube from the incubator or heating block and placing it on ice. 
	NOTE: After allowing the &#946;-elimination reaction to proceed for 18 hr, the desalting process must be performed within the same day.</li>
</ol>
3. Desalting on Cation-exchange Chromatography
<ol>
	<li>While maintaining the sample tube on ice to prevent excessive heat formation, slowly add 10% acetic acid (AcOH) dropwise to neutralize the base. Gently vortex the sample tube between additions of AcOH. Be careful to add acid slowly and drop wise as the addition of acid will produce a &#8220;volcano&#8221; of bubbles. Centrifuge the tube to eliminate bubbles and prevent spillover, if necessary.
	<ol>
		<li>In order to remove the large amount of sodium in the reaction mixture, pass the reaction mixture through a small cation-exchange column (Dowex or AG-50W-X8 resin, H+ form).</li>
		<li>Wash the cation exchange resin with 1 M NaOH and 1 M HCl prior to use in order to remove contaminants that interfere with MS analysis, even if the resin is of analytical grade.</li>
		<li>Soak the resin in 1 M NaOH and remove solution by decantation. Add de-ionized water, remove water, then add 1 M HCl.</li>
	</ol>
	</li>
	<li>Repeat these steps until the solution above the resin is colorless. Wash the cleaned resin with deionized water and store in 5% AcOH at 4 &#176;C.</li>
	<li>To make a small glass column, scratch and break the tip of a Pasteur glass pipette using a ceramic cutter so that the taper of the pipette tip is approximately 1 cm long. Tease apart a plug of glass wool and push towards the tip forming a support for the resin bed. Secure the pipette column with a clamp or clothespin and place over a glass test tube.</li>
	<li>Wash the empty pipette with 1 ml of methanol (MeOH) and 3 ml of 5% AcOH. Swirl H+ Dowex or AG50 cation exchange resin slurry stored in 5% AcOH and transfer enough of the slurry to produce a 1 ml bed volume in the Pasteur pipette column.</li>
	<li>Rinse the column using five volumes of 5% AcOH. Check flow through for the appearance of resin particles and do not use the column if resin is detected.</li>
	<li>Place column over a new screw top glass tube (16 x 125 mm) and load sample. Collect the flow through and elute glycans with at least 3 volumes of 5% AcOH into the same tube.</li>
	<li>Cover the tube with Parafilm and make tiny holes with a needle. Place the sample tube at -80 &#176;C or on dry ice. Once frozen, dry the sample by lyophilization. Store the dried material at -20 &#176;C until use.</li>
</ol>
4. Borate Removal
<ol>
	<li>Prepare 10% AcOH in MeOH. Take 10 ml of glacial acetic acid and adjust volume to 100 ml with methanol. Store this reagent in a glass bottle with Teflon-lined cap for up to 6 months.</li>
	<li>Add 300 &#181;l of 10% AcOH in MeOH to the dried, lyophilized sample tube and vortex. Remove the resulting trimethyl borate by evaporation under a N2 stream. 
	NOTE: Mixing borate salts with methanol under acidic conditions produces trimethyl borate which is a volatile compound.</li>
	<li>Dry under N2 stream at 37 &#176;C. After drying, add another aliquot of AcOH in MeOH as described in step 4.2. Dry the liquid for 5 min under nitrogen (N2) stream at 37 &#176;C.
	<ol>
		<li>Repeat resuspension and drying at least 3 times. Store the dried material at -20 &#176;C until use.</li>
	</ol>
	</li>
</ol>
5. C18 Clean-up
<ol>
	<li>Equilibrate a C18 cartridge column (size 1 ml, 100 mg resin) using three volumes of ACN and five volumes of 5% AcOH. Accelerate passage of the equilibrating solutions by application of mild positive air pressure.</li>
	<li>Add 500 &#181;l of 5% AcOH to the desalted and borate-free sample tube and dissolve by vortex.</li>
	<li>Load resuspended sample onto the equilibrated C18 column and collect the flow through into a glass screw cap tube (13 x 100 mm). Elute O-glycans with 3 ml of 5% AcOH into the same tube.</li>
	<li>Parafilm the tube, make small holes with a small needle or use loosely closed PTFE-lined cap to cover the tube, and freeze the sample at -80 &#176;C or on dry ice. Remove solvent by lyophilization. Store the dried sample at -20 &#176;C until use.</li>
</ol>
6. Base Preparation for Permethylation
NOTE: In order to achieve robust permethylation, prepare NaOH slurry fresh. All glassware used for permethylation reactions should be extensively cleaned.
<ol>
	<li>To prepare the base reagent for permethylation, add 400 &#181;l of 50% NaOH to a clean glass screw-top tube (13 x 100 mm). Add 800 &#181;l of anhydrous MeOH and vortex.</li>
	<li>Add 4 ml of anhydrous dimethyl sulfoxide (DMSO) and vortex to generate a white precipitate.</li>
	<li>Centrifuge at 600 x g for 1 min to pellet the precipitate. Pipette off supernatant and discard. Add an additional 4 ml of anhydrous DMSO to the pellet. Vortex.</li>
	<li>Repeat step 6.2 and 6.3 a minimum of three more times or until no white precipitate forms.</li>
	<li>Dissolve the pelleted base in 3 ml anhydrous DMSO and gently mix by pipetting up-and-down with a clean Pasteur pipette.</li>
	<li>Use the base immediately for the following permethylation reaction as it cannot be stored.</li>
</ol>
7. Permethylation of Released Glycans
<ol>
	<li>Add 200 &#181;l of anhydrous DMSO to the C18-purified sample and vortex or sonicate to resuspend.</li>
	<li>Add 300 &#181;l of the resuspended base slurry to the sample and immediately add 100 &#181;l of iodomethane (MeI). Seal tube with Teflon-lined cap and vigorously mix for 5 min by vortex.</li>
	<li>To stop the permethylation reaction, add 2 ml of 5% AcOH on ice and vortex. Pipette solution up and down 5 times to reduce the remaining volume of MeI by evaporation. 
	NOTE: The AcOH neutralizes the solution and removal of MeI increases the extraction efficiency of sulfated glycans into the water phase during the phase-partition.</li>
	<li>Add 2 ml of dichloromethane (DCM) and vortex. Centrifuge at 600 x g for 1 min at room temperature to separate the aqueous and organic phases.</li>
	<li>Transfer the top layer (aqueous phase that contains the majority of permethylated sulfated glycans) into a new glass tube (13 x 100 mm).</li>
	<li>Add 2 ml of H2O to the organic phase and vortex. Centrifuge at 600 x g for 1 min to separate the aqueous and organic phases and combine this second aqueous phase with the first aqueous phase (step 7.5).</li>
	<li>Repeat step 7.6 and 7.5 three more times, but there is no need to save the aqueous phases resulting from the subsequent partitions.</li>
	<li>Remove the final top layer (aqueous phase) as much as possible from the bottom layer (organic phase) and transfer the bottom layer (organic phase) into a separate new glass tube using clean Pasteur pipette.</li>
	<li>Dry the organic phase under N2 stream at 42 &#176;C.</li>
	<li>Cover the tube with Parafilm and store at -20 &#176;C until use.</li>
</ol>
8. C18 Clean-up of Permethylated Sulfated O-glycans from the Aqueous Phase
<ol>
	<li>Equilibrate a C18 cartridge column with three volumes of ACN and five volumes of 5% AcOH.</li>
	<li>Load the aqueous phases collected and combined from steps 7.5 and 7.6 onto the column.</li>
	<li>Wash the column with 10 ml of H2O for desalting.</li>
	<li>Elute the permethylated sulfated O-glycans into a new glass tube with 2 ml of 50% ACN. Use an additional elution with 85% ACN to enhance recovery of permethylated sulfated O-glycans on larger oligosaccharides.</li>
	<li>Dry eluates under nitrogen stream at 42 &#176;C.</li>
	<li>Store the dried material at -20 &#176;C up to 6 months prior to MS analysis.</li>
</ol>
9. Mass Spectrometry of Permethylated O-glycans
<ol>
	<li>Analyze permethylated O-glycans by direct infusion into an appropriate mass spectrometer using a nanoelectrospray source at a syringe flow rate of 0.40&#8211;0.60 &#181;l/min at 210 &#176;C capillary temperature. For fragmentation by CID in MS/MS and MSn of an ion trap instrument, apply 30&#8211;40% collision energy.</li>
	<li>For negative ion mode, reconstitute permethylated sulfated O-glycans in 50 &#956;l of methanol/2-propanol/1-propanol/13 mM aqueous ammonium acetate (16:3:3:2 by volume). For positive ion mode, reconstitute permethylated neutral/sialylated O-glycans in 50 &#181;l of 1 mM sodium hydroxide in methanol/water (1:1).</li>
	<li>Use the total ion mapping (TIM) and neutral loss scan (NL scan) functionality of the Xcalibur software package version 2.0 for MSn analysis to characterize individual O-glycan structures 13,19.</li>
</ol>

<ol>
	<li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+roles+of+oligosaccharides:+all+of+the+theories+are+correct.">Biological roles of oligosaccharides: all of the theories are correct.</a> Glycobiology. 3 (2), 97-130 (1993).</li><li>Ohtsubo, K., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+in+cellular+mechanisms+of+health+and+disease.">Glycosylation in cellular mechanisms of health and disease.</a> Cell. 126 (5), 855-867 (2006).</li><li>Moremen, K. W., Tiemeyer, M., Nairn, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+protein+glycosylation:+diversity,+synthesis+and+function.">Vertebrate protein glycosylation: diversity, synthesis and function.</a> Nat. Rev. Mol. Cell Biol. 13 (7), 448-462 (2012).</li><li>Lowe, J. B., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+approach+to+Mammalian+glycan+function.">A genetic approach to Mammalian glycan function.</a> Annu. Rev. Biochem. 72, 643-691 (2003).</li><li>Yan, A., Lennarz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+mechanism+of+protein+N-glycosylation.">Unraveling the mechanism of protein N-glycosylation.</a> J. Biol. Chem. 280 (5), 3121-3124 (2005).</li><li>Washburn, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilisation+of+proteomics+datasets+generated+via+multidimensional+protein+identification+technology+(MudPIT).">Utilisation of proteomics datasets generated via multidimensional protein identification technology (MudPIT).</a> Brief Funct. Genomic Proteomic. 3 (3), 280-286 (2004).</li><li>Rosenfeld, J., Capdevielle, J., Guillemot, J. C., Ferrara, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-gel+digestion+of+proteins+for+internal+sequence+analysis+after+one-+or+two-dimensional+gel+electrophoresis.">In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis.</a> Anal. Biochem. 203 (1), 173-179 (1992).</li><li>Hellman, U., Wernstedt, C., Gonez, J., Heldin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+an+'In-Gel'+digestion+procedure+for+the+micropreparation+of+internal+protein+fragments+for+amino+acid+sequencing.">Improvement of an 'In-Gel' digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing.</a> Anal. Biochem. 224 (1), 451-455 (1995).</li><li>Morelle, W., Canis, K., Chirat, F., Faid, V., Michalski, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+mass+spectrometry+for+the+proteomic+analysis+of+glycosylation.">The use of mass spectrometry for the proteomic analysis of glycosylation.</a> Proteomics. 6 (14), 3993-4015 (2006).</li><li>Mortz, E., Sareneva, T., Haebel, S., Julkunen, I., Roepstorff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+characterization+of+glycosylated+interferon-gamma+variants+separated+by+gel+electrophoresis.">Mass spectrometric characterization of glycosylated interferon-gamma variants separated by gel electrophoresis.</a> Electrophoresis. 17 (5), 925-931 (1996).</li><li>Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A., Harvey, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequencing+of+N-linked+oligosaccharides+directly+from+protein+gels:+in-gel+deglycosylation+followed+by+matrix-assisted+laser+desorption/ionization+mass+spectrometry+and+normal-phase+high-performance+liquid+chromatography.">Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography.</a> Anal. Biochem. 250 (1), 82-101 (1997).</li><li>Kuster, B., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18O-labeling+of+N-glycosylation+sites+to+improve+the+identification+of+gel-separated+glycoproteins+using+peptide+mass+mapping+and+database+searching.">18O-labeling of N-glycosylation sites to improve the identification of gel-separated glycoproteins using peptide mass mapping and database searching.</a> Anal. Chem. 71 (7), 1431-1440 (1999).</li><li>Aoki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diversity+of+O-linked+glycans+expressed+during+Drosophila+melanogaster+development+reflects+stage-+and+tissue-specific+requirements+for+cell+signaling.">The diversity of O-linked glycans expressed during Drosophila melanogaster development reflects stage- and tissue-specific requirements for cell signaling.</a> J. Biol. Chem. 283 (44), 30385-30400 (2008).</li><li>Kozak, R. P., Royle, L., Gardner, R. A., Fernandes, D. L., Wuhrer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+peeling+during+the+release+of+O-glycans+by+hydrazinolysis.">Suppression of peeling during the release of O-glycans by hydrazinolysis.</a> Anal. Biochem. 423 (1), 119-128 (2012).</li><li>Schulz, B. L., Packer, N. H., Karlsson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-scale+analysis+of+O-linked+oligosaccharides+from+glycoproteins+and+mucins+separated+by+gel+electrophoresis.">Small-scale analysis of O-linked oligosaccharides from glycoproteins and mucins separated by gel electrophoresis.</a> Anal. Chem. 74 (23), 6088-6097 (2002).</li><li>Thomsson, K. A., Schulz, B. L., Packer, N. H., Karlsson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUC5B+glycosylation+in+human+saliva+reflects+blood+group+and+secretor+status.">MUC5B glycosylation in human saliva reflects blood group and secretor status.</a> Glycobiology. 15 (8), 791-804 (2005).</li><li>Taylor, A. M., Holst, O., Thomas-Oates, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+profiling+of+O-linked+glycans+released+directly+from+glycoproteins+in+gels+using+in-gel+reductive+&#946;-elimination.">Mass spectrometric profiling of O-linked glycans released directly from glycoproteins in gels using in-gel reductive &#946;-elimination.</a> Proteomics. 6 (10), 2936-2946 (2006).</li><li>Anumula, K. R., Taylor, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+procedure+for+preparation+of+partially+methylated+alditol+acetates+from+glycoprotein+carbohydrates.">A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates.</a> Anal. Biochem. 203 (1), 101-108 (1992).</li><li>Aoki, K., Perlman, M., Lim, J. M., Cantu, R., Wells, L., Tiemeyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+developmental+elaboration+of+N-linked+glycan+complexity+in+the+Drosophila+melanogaster+embryo.">Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo.</a> J. Biol. Chem. 282 (12), 9127-9142 (2007).</li><li>Domon, B., Costello, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Systematic+Nomenclature+for+Carbohydrate+Fragmentations+in+Fab-Ms+Ms+Spectra+of+Glycoconjugates.">A Systematic Nomenclature for Carbohydrate Fragmentations in Fab-Ms Ms Spectra of Glycoconjugates.</a> Glycoconj. J. 5 (4), 397-409 (1988).</li><li>Ashline, D., Singh, S., Hanneman, A., Reinhold, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congruent+strategies+for+carbohydrate+sequencing.+1.+Mining+structural+details+by+MSn.">Congruent strategies for carbohydrate sequencing. 1. Mining structural details by MSn.</a> Anal. Chem. 77 (19), 6250-6262 (2005).</li><li>Lapadula, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congruent+strategies+for+carbohydrate+sequencing.+3.+OSCAR:+an+algorithm+for+assigning+oligosaccharide+topology+from+MSn+data.">Congruent strategies for carbohydrate sequencing. 3. OSCAR: an algorithm for assigning oligosaccharide topology from MSn data.</a> Anal. Chem. 77 (19), 6271-6279 (2005).</li><li>Ashline, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+structural+isomers+analyzed+by+sequential+mass+spectrometry.">Carbohydrate structural isomers analyzed by sequential mass spectrometry.</a> Anal. Chem. 79 (10), 3830-3842 (2007).</li><li>Thomsson, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+salivary+mucin+MG1+(MUC5B)+carries+a+repertoire+of+unique+oligosaccharides+that+is+large+and+diverse.">The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and diverse.</a> Glycobiology. 12 (1), 1-14 (2002).</li><li>Yu, S. Y., Wu, S. W., Hsiao, H. H., Khoo, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enabling+techniques+and+strategic+workflow+for+sulfoglycomics+based+on+mass+spectrometry+mapping+and+sequencing+of+permethylated+sulfated+glycans.">Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.</a> Glycobiology. 19 (10), 1136-1149 (2009).</li><li>Yu, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming+mass+spectrometry-based+sulfoglycomic+mapping+for+identification+of+terminal+sulfated+lacdiNAc+glycotope.">Priming mass spectrometry-based sulfoglycomic mapping for identification of terminal sulfated lacdiNAc glycotope.</a> Glycoconj J. 30 (2), 183-194 (2013).</li><li>Kumagai, T., Katoh, T., Nix, D. B., Tiemeyer, M., Aoki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-gel+&#946;-elimination+and+aqueous-organic+partition+for+improved+O-+and+sulfoglycomics.">In-gel &#946;-elimination and aqueous-organic partition for improved O- and sulfoglycomics.</a> Anal. Chem. 85 (18), 8692-8699 (2013).</li></ol>

The authors declare no competing financial interest.

Combining in-gel reductive &#946;-elimination with aqueous-organic extraction enhances the sensitivity and depth of structural data that can be acquired for characterizing sulfated and non-sulfated O-linked glycans harvested from small amounts of mucin-type glycoproteins resolved from other proteins by SDS-PAGE. The essential advances of the technical approaches presented in this study are: (a) facile removal of gel derived contaminants by simple washing steps; (b) quantitative recovery of permethylated sulfoglycans in the aqueous phase following a rapid water-DCM partition. The workflow described here dramatically improves the sensitivity of detection for sulfated and non-sulfated glycans released from glycoproteins expressed at physiological levels in biological samples. 
While the methods described here are robust and highly reproducible when applied with careful attention to standard analytic practices, a few considerations regarding possible sources of contamination are worth a brief mention. Ensuring that all glassware is exceptionally clean and free of dust and detergent is critical. Upon noticing contaminant peaks in MS spectra, all glassware should be washed thoroughly and rinsed with MeOH prior to final drying. Another source of contamination is the use of rubber-lined screw caps in place of PTFE-lined caps. The lack of organic chemical resistance characteristic of the rubber lining results in samples contaminated with an interfering polymeric compound. Although this protocol was specifically designed with the intention of utilizing MSn analysis by NSI-MS on an ion trap instrument, other types of mass spectrometers, such as MALDI-TOF/TOF can be also applied to dissecting sulfoglycoforms in biological materials using the preparative methods described here.
In comparison to other methodologies currently in use for in-gel glycan release and analysis, the techniques described here do not require HPLC separation prior to MS analysis and are applicable to permethylated glycans, producing greater structural characterization than can be extracted from non-derivatized samples. Moreover, the methods described here are capable of separating permethylated forms of sulfated O-glycans from non-sulfated neutral and sialylated O-glycans by a simple phase partition. The optimized sample clean-up and simple phase partition methodologies minimize sample manipulation and losses that can occur through chromatography steps. Methods requiring greater sample handling may lead to an underestimate of sulfoglycan diversity due to losses during work-up. Furthermore, detection of permethylated sulfated glycans in negative mode is significantly less sensitive than detection of permethylated glycans in positive mode. Desulfation of permethylated sulfoglycans by solvolysis specifically removes sulfate groups, leaving naked hydroxyls that can be subsequently permethylated with deuterated methyl iodide, increasing MS-signal intensities approximately 5-fold and providing a unique isotopic mass tag at the site of sulfation25.

Glycosylation is an essential protein post-translational modification, contributing to organismal physiology, tissue pathology, and cellular recognition 1-3. Despite major advances in analytical glycoscience, characterizing the complete diversity of glycans on a specific protein remains an extremely challenging task, especially on proteins isolated from primary biological sources. Nonetheless, the microheterogeneity of glycoprotein glycans frequently affects functional interactions with other proteins. Therefore, characterization of glycan diversity is essential for understanding the physiological significance of cellular and tissue glycosylation 4,5. In order to understand the contribution of glycoprotein glycosylation to tissue physiology and pathophysiology, robust, sensitive, and comprehensive glycomic analytical techniques have become increasingly important. In proteomic analysis, protein identifications are generally achieved by LC-MS/MS analysis of tryptic peptides 6. Protein digestion can be carried out using a purified protein or proteins resolved by SDS-PAGE following in-gel digestion with proteases such as trypsin 7-9. Pre-enrichment of the protein mixture by SDS-PAGE enhances the depth and accuracy of protein ID. The development of analogous strategies for glycomic analysis of glycoprotein glycosylation lies at the forefront of glycoscience.
The two major classes of glycans are attached to protein backbones through either N-linkage or O-linkage. N-linked glycans are attached to asparagine (Asn) residues found as part of a sequon defined as Asn-X-Ser/Thr/Cys (X is any amino acid except proline), and can be released by enzymatic digestion with peptide-N-glycanase (PNGaseF or A), either in solution, in-gel, or on-blot 10-12. O-linked glycans are mainly attached to serine (Ser) or threonine (Thr) residues. However, only one enzyme has been identified that is capable of releasing O-linked glycans from glycoprotein and it has an extremely limited glycan specificity, releasing only the simplest O-linked glycans. Chemical release strategies remain the method of choice for comprehensive release of O-linked glycans from glycoproteins. Reductive or non-reductive &#946;-elimination, or hydrazinolysis are well-characterized chemical release techniques and are currently the most commonly used approaches for releasing O-linked glycans from glycoproteins 13,14. Although reductive &#946;-elimination has been used to release O-linked glycans from glycoproteins separated by SDS-PAGE, previous approaches required HPLC separation for subsequent analysis 15-17.
Multidimensional MS (MSn) analysis currently provides the richest source of structural data for characterizing glycans released from glycoproteins isolated in the amounts expected from most biological sources. The depth of MS-based structural characterization is greatly facilitated by permethylating the released glycans prior to their analysis. Permethylation enhances ionization and tends to equalize molar signal responses across a broad range of glycan structures 18,19. In addition, permethylation unambiguously tags terminal and substituted monosaccharide moieties with distinctive masses, thereby enhancing structural elucidation 20-23. For example, acidic glycans are generally difficult to detect as non-permethylated species by MS. Although acidic glycans can be detected in negative ion mode by MS, it is impossible to detect both acidic and neutral glycans in the same ion mode. A major advantage of glycan permethylation is that all of the free hydroxyl groups (OH) on a glycan&#8217;s monosaccharide substituents will be capped with a methyl group (OCH3 or OMe), thus a sialylated glycan&#8217;s charges are neutralized, making them as detectable as permethylated neutral (asialo) glycans. However, the hydroxyls of sulfate moieties on sulfoglycans are resistant to permethylation, resulting in retention of anionic charge, which suppresses ionization and decreases sensitivity. This suppression currently prevents comprehensive glycomic analysis of very complex glycoproteins such as mucins, which carry a high abundance of sulfated glycans 24-26.
Recent reports on purifying sulfated glycans used charged, reverse-phase chromatography to purify and separate permethylated glycans prior to MALDI analysis. This method relies on complete separation of sulfated and non-sulfated glycans using different mobile phases for elution, which we have found to be less stringent than organic phase partitioning. Therefore, new techniques suitable for the detection and enrichment of sulfoglycans are presented here. These techniques allow for the quantitative recovery of sulfated glycans in the aqueous phase following water:DCM (dichloromethane) extraction, which is routinely performed at the end of glycan permethylation reactions 27. Importantly, this robust separation of permethylated sulfoglycans from a mixture of permethylated non-sulfated glycans concomitantly enriches for charged species while also simplifying MS2 fragmentation patterns. A comprehensive protocol for improved in-gel O-linked glycan analysis is also presented. The improved protocol enhances glycan recovery, increases the structural information obtainable through MSn analysis of permethylated glycans, and improves the sensitivity of sulfoglycomic analyses applied to essential glycoproteins isolated from biological sources.
This protocol is intended for O-linked glycan analysis of whole glycoprotein extracts or of a specific glycoprotein of interest resolved by SDS-PAGE and is composed of three experimental procedures; A) gel clean-up, B) in-gel reductive &#946;-elimination, and C) glycan permethylation. The goal is to obtain comprehensive O-linked glycomic data for glycoproteins harvested from primary sources of biological interest (Figure 1). Glycoproteins separated by SDS-PAGE are visualized by staining and bands of interest are excised and the resulting gel band is sliced into small pieces. The gel pieces are destained and subjected to ethyl acetate washes to remove gel contaminants (Figure 2A). Glycan release is achieved by in-gel reductive &#946;-elimination (Figure 2B) and the released glycans are permethylated. Aqueous-organic extraction following permethylation quantitatively partitions the anionic sulfated glycans away from non-sulfated neutral glycans (Figure 2C). In-gel reductive &#946;-elimination coupled to aqueous-organic extraction enables the characterization of O-linked glycans and sulfoglycans released from small amounts of glycoprotein separated by SDS-PAGE. The strategic overview is summarized in Figure 1 and the details are shown in Figure 2. In addition, a portion of the destained and washed gel pieces can be used for protein ID by standard LC-MS/MS proteomic techniques.

<table><tbody><tr><td>Water, deionized water</td><td>Sigma-Aldrich&amp;nbsp;</td><td>270733-4L</td><td>CHROMASOLV, for HPLC</td></tr><tr><td>Acetic acid, Glacial</td><td>Fisher</td><td>A38-500</td><td>Certified ACS &amp;ge;99.7% w/w</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>34860-4L-R</td><td>CHROMASOLV, for HPLC, &amp;ge;99.9%</td></tr><tr><td>Ammonium bicarbonate</td><td>Fluka</td><td>09830-500G</td><td>BioUltra, &amp;ge;99.5% (T), Step 1</td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich&amp;nbsp;</td><td>34998-4L</td><td>CHROMASOLV Plus, for HPLC, &amp;ge;99.9%, Step 1</td></tr><tr><td>Ethyl acetate</td><td>Fluka</td><td>34972-1L-R</td><td>LC-MS CHROMASOLV, Step 1</td></tr><tr><td>Sodium borohydride</td><td>Aldrich</td><td>213462-25G</td><td>ReagentPlus, 99%, Step 2</td></tr><tr><td>Iodomethane</td><td>Sigma-Aldrich&amp;nbsp;</td><td>289566-100G</td><td>ReagentPlus, 99.5%,&amp;nbsp; Step 6.&amp;nbsp; Store at 4 &amp;deg;C until use.&amp;nbsp; Sit at room temperature before use.</td></tr><tr><td>Sodium hydroxide solution, 50% w/w</td><td>Fisher</td><td>SS254-1</td><td>Certified, Step 6</td></tr><tr><td>Dimethyl sulfoxide, anhydrous</td><td>Sigma-Aldrich&amp;nbsp;</td><td>276855-1L</td><td>Anhydrous, &amp;ge;99.9%, Step 6</td></tr><tr><td>Methanol, anhydrous</td><td>Sigma-Aldrich&amp;nbsp;</td><td>322415-100ML</td><td>Anhydrous, 99.8%, Step 6</td></tr><tr><td>Dichloromethane</td><td>Sigma-Aldrich&amp;nbsp;</td><td>34856-4L</td><td>CHROMASOL&amp;reg;, for HPLC, &amp;ge;99.8%, contains amylene as stabilizer, Step 7</td></tr><tr><td>Dowex 50WX8 hydrogen formhydrogen form 100-200&amp;nbsp;mesh</td><td>Sigma-Aldrich&amp;nbsp;</td><td>217506-500G</td><td>Step 3</td></tr><tr><td>AG 50W-X8 Resin&amp;nbsp;</td><td>Bio-Rad</td><td>142-1441</td><td>Step 3</td></tr><tr><td>BAKERBOND spe 1 ml x 100 mg Solid Phase Extraction Column, PP, Octadecyl (C18) Reverse Phase</td><td>JT Baker</td><td>JT-7020-01</td><td>Step 5 and 8</td></tr><tr><td>7.5% Mini-PROTEAN TGX Precast Gel&amp;nbsp;</td><td>Bio-Rad</td><td></td><td>Step 1</td></tr><tr><td>Bio-Safe Coomassie Stain&amp;nbsp;</td><td>Bio-Rad</td><td>161-0786</td><td>G-250, Step 1</td></tr><tr><td>Silver Stain Kit for Mass Spectrometry</td><td>Pierce</td><td>24600</td><td>Step 1</td></tr><tr><td>Oligosaccharides Kit (Maltotriose, Dp3: R474140)</td><td>Supelco&amp;nbsp;</td><td>47265</td><td></td></tr><tr><td>Oligosaccharides Kit (Maltotetraose, Dp4: R474135)</td><td>Supelco&amp;nbsp;</td><td>47265</td><td></td></tr><tr><td>Disposable Pasteur Pipets, Glass, Short Tip</td><td>VWR</td><td>14673-010</td><td>Wash before use</td></tr><tr><td>PYREX 13 x 100 mm Disposable Round Bottom Threaded Culture Tubes</td><td>Corning</td><td>99447-13</td><td>Wash before use</td></tr><tr><td>PYREX 16 x 125 mm Disposable Round Bottom Threaded Culture Tubes</td><td>Corning</td><td>99447-16</td><td>Wash before use</td></tr><tr><td>Phenolic Caps/Closures with PTFE-Faced Rubber Liner</td><td>Kimble</td><td>45066C-13</td><td>Wash before use</td></tr><tr><td>Phenolic Caps/Closures with PTFE-Faced Rubber Liner</td><td>Kimble</td><td>45066C-15</td><td>Wash before use</td></tr><tr><td>Hamilton HPLC syringe</td><td>HAMILTON</td><td>81265</td><td>volume 500&amp;nbsp;&amp;mu;l, needle size 22 G</td></tr><tr><td>Hamilton HPLC syringe</td><td>HAMILTON</td><td>81165</td><td>volume 250&amp;nbsp;&amp;mu;l, needle size 22 G</td></tr><tr><td>Hamilton HPLC syringe</td><td>HAMILTON</td><td>81065</td><td>volume 100&amp;nbsp;&amp;mu;l, needle size 22s G</td></tr><tr><td>Hamilton HPLC syringe</td><td>HAMILTON</td><td>80965</td><td>volume 50&amp;nbsp;&amp;mu;l, needle size 22s G</td></tr><tr><td>Hamilton Calibrated Syringes</td><td>HAMILTON</td><td>80300</td><td>volume 10&amp;nbsp;&amp;mu;l, needle size 26s G</td></tr><tr><td>Petri Dish Glass 100 mm x 15mm</td><td>GSC INTERNATIONAL INC</td><td>1500-4</td><td>Wash before use, Step 1</td></tr><tr><td>Bard-Parker Surgical Blades</td><td>Fisher</td><td>371310</td><td>Step 1</td></tr><tr><td>Reacti-Therm Heating/Stirring Module</td><td>Pierce</td><td>18870</td><td>Step 1, 4 and 7</td></tr><tr><td>Heating Blocks</td><td>Fisher</td><td>125D</td><td>Step 2</td></tr><tr><td>Pyrex fiber glass wool borosilicate pore size 8&amp;nbsp;&amp;mu;m</td><td>Aldrich</td><td>CLS3950</td><td>Step 3</td></tr><tr><td>Fused Silica CutterTubing cutter</td><td>alltech</td><td>3194</td><td>Step 3</td></tr><tr><td>Multi-tube vortexers&amp;nbsp;</td><td>VWR</td><td>444-7063</td><td>Step 6</td></tr><tr><td>Lyophilizer 25EL Freezemobile</td><td>Virtis</td><td>25EL</td><td></td></tr><tr><td>Centrifuge</td><td>VWR</td><td>Clinical 50</td><td></td></tr><tr><td>LTQ Orbitrap Discovery</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr></tbody></table>

improved in-gel reductive &#946;-elimination for comprehensive o-linked and sulfo-glycomics by mass spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Effect of Ethyl Acetate Treatment Prior to In-gel Reductive &#946;-Elimination
A representative mass spectrum of permethylated O-linked glycan samples released from bovine mucin using in-gel reductive &#946;-elimination is shown in Figure 3. The EtOAc wash of the gel pieces effectively removes SDS and polyacryl contaminants which interfere with subsequent MS analysis 27.
<img alt="Figure 3" src="/files/ftp_upload/51840/51840fig3highres.jpg" /> 
Figure 3. Ethyl acetate wash of polyacrylamide gel piece prior to in-gel reductive &#946;-elimination enhances detection of released glycans. (A) Without wash with ethyl acetate, the mass spectrum of permethylated O-linked glycans released by in-gel &#946;-elimination from bovine submaxillary mucin is dominated by an abundance of a polydisperse contaminant, which almost completely obscures the MS signals associated with O-linked glycans. (B) Washing the gel piece with ethyl acetate eliminates the contaminant peaks, allowing sensitive detection of glycans. Modified from Kumagai25.
Recovery of O-linked Glycan is Greater from Small Gel Slices
O-linked glycans were released from bovine submaxillary mucin by in-gel reductive &#946;-elimination using gel pieces that were either sliced small (~2 x 2 mm) or large (~5 x 5 mm). Gel pieces smaller than ~2 x 2 mm were not efficiently recovered through the washing steps. Following permethylation, a known amount of a permethylated external glycan standard (maltotri- and maltotetrasaccharide, Dp3 and Dp4) was added to each to facilitate quantification of glycan recovery 27. The recovery of O-linked glycans from small gel pieces was almost 10-fold greater than from large gel slices (Figure 4).
<img alt="Figure 4" fo:content-width="2.5in" src="/files/ftp_upload/51840/51840fig4highres.jpg" width="250px" /> 
Figure 4. Glycan recovery from small gel pieces is more efficient than from larger pieces. Small pieces (~2 x 2 mm) yielded more glycan than larger cubes (~5 x 5 mm). Bars show relative signal intensities for the sum of all O-linked glycan structures normalized to the signal detected for an external standard (maltotrisaccharide Dp3, set to 100%), which was added to the released glycan before MS analysis. Values are mean standard deviation for n=3. Modified from Kumagai K25.
Enrichment of Sulfoglycans by Phase Partition 
A permethylated sulfated glycan standard (sulfo-Lea) was completely recovered in the aqueous phase. Permethylated sialylated glycans released from the mucin glycoprotein partitioned into the DCM phase (Figure 5). The recovery of each permethylated glycans by aqueous-organic partition was comparable to that of the previously characterized C18 Sep-Pak clean-up method 27. The efficiency and simplicity of the phase partition method greatly facilitates sample throughput and subsequent analytic approaches.
<img alt="Figure 5" src="/files/ftp_upload/51840/51840fig5highres.jpg" /> 
Figure 5. Differential recovery of sulfo- and sialo-glycans by phase partition. Bovine mucin glycoprotein was spiked with a known amount of a sulfo-glycan standard (sulfo-Lea) before being subjected to reductive &#946;-elimination. Released glycans were permethylated and the permethylation reaction was adjusted to 1:1:water:DCM. The resulting organic and aqueous phases were separated and analyzed by MS. Glycan recovery was quantified relative to permethylated external glycan standards, which were spiked into the sample before MS analysis. Glycan recoveries into the organic (DCM) and aqueous (Water) phases are shown relative to the external standard, which was set to 100%. Sulfoglycans were undetectable in the organic phase but quantitatively recovered in the aqueous phase. Results represent the mean &#177; S.E. of three independent experiments. Modified from Kumagai25.
Application to In-depth Proteomic and Glycomic Analyses in Biological Samples
Human saliva proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (G-250). A high molecular weight protein at MW ~600 kDa was excised from the gel and a portion of the gel piece was subjected to in-gel tryptic digestion and LC-MS/MS-based proteomic analysis, which identified this band as MUC5B 16,24. The remainder of the gel piece was subjected to in-gel reductive &#946;-elimination for O-glycan analysis 27. Following permethylation and aqueous-organic phase partition (Water:DCM, 1:1), permethylated glycans in the aqueous and organic phases were analyzed by NSI-MS. Non-sulfated permethylated O-glycans were recovered from the organic phase and all of the permethylated sulfoglycans were recovered from the aqueous phase, which facilitates the identification and characterization of nearly isobaric sulfated and non-sulfated glycans, e.g., Fuc1Hex3HexNAc2GalNAc-ol (m/z=1606.8, [M+Na]+) and (SO3)1NeuAc1Fuc1Hex2HexNAc1GalNAc-ol (m/z=1606.7, [M+2Na-H]+) differ by only 0.1 mass units and would be difficult to resolve without physically separating the two species by phase partition (Figure 6).
<img alt="Figure 6" src="/files/ftp_upload/51840/51840fig6highres.jpg" width="700px" /> 
Figure 6. Detection of isobaric complexity in neutral and sulfo-glycans separated by aqueous-organic partition. MS2 fragmentation patterns obtained from parent ions detected by total ion mapping (TIM) analysis of permethylated O-glycans released from human salivary mucin by in-gel &#946;-elimination and water:DCM partition following permethylation. (A) MS2 from TIM analysis of DCM phase for a 2.8 mass unit window around m/z = 1608. (B) The MS2 spectrum of the same mass window for TIM analysis of the water phase. In the DCM-phase, the major fragment ions correspond to loss of Hex1-O (m/z 1370.8), Fuc1Hex3HexNAc2+Na (1331.8), loss of Fuc1Hex1-O (1196.7), loss of Hex1HexNAc1 (1143.6), loss of Fuc1Hex1HexNAc1 (969.5), loss of Fuc1Hex2HexNAc1-O (747.4), Fuc1Hex1HexNAc1+Na (660.4), and Hex1HexNAc1+Na (486.3). Based on these fragment ions, a mixture of non-sulfated structures with a composition of Fuc1Hex3HexNAc2GalNAc-ol is proposed as shown to the right of the spectrum. In contrast, the major fragment ions for the water phase are loss of SO3Na (1486.8), loss of NeuAc (1231.5), loss of Fuc1Hex1-O (1196.7), loss of combination of NeuAc and SO3Na (1111.8), and loss of NeuAc1Hex1-O (1009.4). A mixture of sulfated structures with a composition of (SO3-)1NeuAc1Fuc1Hex2HexNAc1GalNAc-ol is proposed. Without physical separation of the neutral and sulfoglycans by phase partition, interpreting MS2 spectra of such mixtures is significantly more challenging. Modified from Kumagai25. <a href="/files/ftp_upload/51840/51840fig6highres.jpg" target="_blank">Please click here to view a larger version of the figure.</a>

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Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

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Nucleophilic Substitution and Elimination Reactions of Alkyl Halides

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Video: Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Immunology and Infection

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture

The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a post-translational modification. Thus, it is important to be able to study this modification in living cells. A simple method to analyze these cysteine-stabilized multimeric complexes is through a two-step method of non-reducing SDS-PAGE analysis and formaldehyde cross-linking. This two-step method is advantageous as the first step to uncovering multimeric complexes stabilized by disulfide linkages due to its technical ease and low cost of operation. Here, the human bone osteosarcoma cell line U-2 OS is used to illustrate this method by specifically analyzing the nuclear isoform of dUTPase.

Disulfide linkages have long been known to stabilize the structure of many proteins. A simple method to analyze multimeric complexes stabilized by these linkages is through non-reducing SDS-PAGE analysis. Here, this method is illustrated by analyzing the nuclear isoform of dUTPase from the human bone osteosarcoma cell line U-2 OS.

The structures of many proteins are stabilized through covalent disulfide linkages. In recent work, this bond has also been classified as a ...

Developmental Biology

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. 
The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed ...

Biochemistry

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Bifidobacteria possess a unique genomic capability for N-glycan cleavage. Recombinantly producing these enzymes would be a promising novel tool to release bioactive N-glycans from glycoprotein-rich substrates such as colostrum.

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, ...

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal infectivity of pathogenic species. Due to this, there is increasing interest in characterizing bacterial glycosylation and a need for high-throughput analytical tools to identify these events. Although bottom-up proteomics readily enables the generation of rich glycopeptide data, the breadth and diversity of glycans observed in prokaryotic species make the identification of bacterial glycosylation events extremely challenging.
Traditionally, the manual determination of glycan compositions within bacterial proteomic datasets made this a largely bespoke analysis restricted to field-specific experts. Recently, open searching-based approaches have emerged as a powerful alternative for the identification of unknown modifications. By analyzing the frequency of unique modifications observed on peptide sequences, open searching techniques allow the identification of common glycans attached to peptides within complex samples. This article presents a streamlined workflow for the interpretation and analysis of glycoproteomic data, demonstrating how open searching techniques can be used to identify bacterial glycopeptides without prior knowledge of the glycan compositions.
Using this approach, glycopeptides within samples can rapidly be identified to understand glycosylation differences. Using Acinetobacter baumannii as a model, these approaches enable the comparison of glycan compositions between strains and the identification of novel glycoproteins. Taken together, this work demonstrates the versatility of open database-searching techniques for the identification of bacterial glycosylation, making the characterization of these highly diverse glycoproteomes easier than ever before.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal ...

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

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.

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 ...

Cancer Research

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

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