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>

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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 t...

グリコシル化は、生物生理学、組織病 ​​理学、および細胞認識1-3に貢献し、必須タンパク質の翻訳後修飾である。分析糖鎖の主要な進歩にもかかわらず、特定のタンパク質上のグリカンの完全な多様性を特徴づけることは、特に主要な生物学的供給源から単離されたタンパク質に、極めて困難な課題である。それにもかかわらず、糖タンパク質グリカンの微小不均一性は、頻繁に他のタンパク質との機能的相互作用に影響を与えます。したがって、グリカン多様性の特徴付けは、細胞および組織のグリコシル4,5の生理的意義を理解するために不可欠である。組織の生理学および病態生理学、堅牢敏感な、包括的なglycomic分析技術の糖タンパク質のグリコシル化の寄与を理解するためにますます重要になってきた。プロテオーム解析では、タンパク質同定は、一般的LC-MSにより達成されるトリプシンペプチド6の/ MS分析。タンパク質消化は、7-9トリプシンなどのプロテアーゼによるゲル内消化後のSDS-PAGEによって分離精製されたタンパク質またはタンパク質を用いて行うことができる。 SDS-PAGEによるタンパク質混合物の予備濃縮は、タンパク質IDの深さと正確さを向上させます。糖タンパク質グリコシル化のglycomic分析のための類似の戦略の開発は、糖質科学の最前線に位置しています。 グリカンの2つの主要なクラスは、N-連結またはO-連結のいずれかを介してタンパク質主鎖に結合している。 N結合型グリカンは、アスパラギンに結合しているアスパラギン（Asn）のAsn-X-Ser / Thr / Cysをとして定義シークの一部として見出される残基は、（Xはプロリンを除く任意のアミノ酸である）、そしてペプチド- Nによる酵素消化により放出させることができる-glycanase（PNGアーゼFまたはA）、いずれの溶液中で、ゲル内、またはオンブロット10-12。 O結合型グリカンは主にセリン（セリ​​ン）またはトレオニン（スレオニン）残基に結合している。ただし、1つの酵素は、IDENされている糖タンパク質のO-結合グリカンを放出することが可能であり、それは唯一の最も単純なO結合型グリカンを放出する、非常に限られたグリカン特異性を有するtified。化学物質放出戦略は、糖タンパク質のO-結合グリカンの包括的放出のための選択の方法のままである。還元的または非還元的β脱離、またはヒドラジンのよく特徴付けられた化学物質放出技術であり、現在糖タンパク質13,14からのO結合グリカンを放出するための最も一般的に使用されるアプローチである。還元的β脱離は、SDS-PAGEによって分離された糖タンパク質のO-結合グリカンを放出するために使用されてきたが、従来のアプローチは、その後の分析のために15〜17 HPLC分離を必要とした。 多次元MS（MS n）の分析では、現在、ほとんどの生物学的情報源から見込額で単離した糖タンパク質から放出されたグリカンを特徴付けるための構造データの最も豊富なソースを提供します。 MSの深さベースの構造的特徴が大幅に先立って彼らの分析にリリースグリカンをpermethylatingによって促進される。完全メチル化は、イオン化を強化し、グリカン構造18,19の幅広い分子のシグナル応答を等化する傾向がある。また、過メチル化は、明確にすることにより構造の解明20-23を高め、独特の大衆を持つ端末および置換単糖部分をタグ付けします。例えば、酸性グリカンはMSにより非過メチル化種として検出することが一般的に困難である。酸性グリカンMS負イオンモードで検出することができるが、同じイオンモードの両方で、酸性および中性グリカンを検出することは不可能である。グリカンの完全メチル化の主要な利点は、完全メチル化のように、グリカンの単糖置換基上の遊離ヒドロキシル基（OH）の全てがそれらのように検出可能にメチル基（OCH 3またはOMeで）、このようにしてシアル化グリカンの電荷が中和されるでキャップされることであるニュートラル（アジアLO）グリカン。しかし、sulfoglycans上の硫酸残基のヒドロキシルイオン化を抑制し、感度を低下させるアニオン電荷の保持をもたらす、完全メチル化に耐性がある。この抑制は、現在、そのような硫酸化グリカン24-26の高い存在を運ぶムチン、など非常に複雑な糖タンパク質の包括的なglycomic分析を防ぎます。 浄化硫酸化グリカン上の最近の報告では、逆相精製するクロマトグラフィーおよびMALDI分析の前に別々の過メチル化グリカン、充電に使用。この方法では、我々は、有機相分配よりもあまり厳密であることがわかってきた溶出のための異なる移動相を使用した硫酸化および非硫酸化グリカンの完全な分離に依存している。したがって、sulfoglycansの検出および濃縮のために適した新しい技術がここに提示されている。これらの技術は、水以下の水相中の硫酸化糖鎖の定量的回収を可能に：DCM（ジクロロメタン）日常グリカン過メチル化反応27の最後に行われ、抽出、。また、MS 2フラグメンテーションパターンを簡素化しながら、重要なことは、完全メチル化非硫酸化グリカンの混合物からの完全メチルsulfoglycansのこの堅牢な分離は、付随的に帯電した種のために豊かにします。改善されたゲル内O-結合グリカン分析のための総合的なプロトコルも提示されている。改良されたプロトコルがグリカンの回復を高め、過メチル化グリカンのMS n分析を通じて得られる構造情報を増加させ、生物学的な供給源から単離さ不可欠糖タンパク質に適用sulfoglycomic分析の感度を向上させます。 このプロトコルは、全体の糖タンパク質抽出物のO結合型グリカン分析のために、またはSDS-PAGEにより分離し、目的の特定の糖タンパク質のことを意図しており、3つの実験の手順で構成されている。 A）は、ゲルクリーンアップ、B）は、ゲル内還元的β脱離、およびC）糖鎖permethy設置と。目標は、生物学的に関心のある主要な供給源から採取された糖タンパク質のための総合的なO結合型glycomicデータ（ 図1）を得ることである。 SDS-PAGEによって分離された糖タンパク質を染色することによって視覚化され、目的のバンドを切り出し、得られたゲルのバンドを小片にスライスされる。ゲル片を脱染色し、ゲルの汚染物質（ 図2A）を除去し、酢酸エチルでの洗浄に供される。グリカン放出は、ゲル内還元的β脱離（ 図2B）によって達成され、遊離されたグリカンを完全メチル化されている。水性有機抽出後に過メチル化を定量的に離れて非硫酸ニュートラルグリカン（ 図2C）からのアニオン性硫酸化グリカンを仕切る。水性 - 有機溶​​媒抽出に結合されたゲル内還元的β脱離は、SDS-PAGEで分離した糖タンパク質の少量から放出されたO結合型グリカンとsulfoglycansの特徴付けを可能にします。戦略的な概要はsummariです図1および詳細におけるZEDは、 図2に示されている。また、脱染色し、洗浄し、ゲル片の部分は、通常のLC-MS / MSプロテオミクス技術によるタンパク質のIDのために使用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="812">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Material/ Equipment</td>
			<td style="width:155px;">Company</td>
			<td style="width:124px;">Catalog Number</td>
			<td style="width:303px;">Comments/Description</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:231px;">Water, deionized water</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>270733-4L</td>
			<td style="width:303px;">CHROMASOLV, for HPLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Acetic acid, Glacial</td>
			<td style="width:155px;">Fisher</td>
			<td>A38-500</td>
			<td style="width:303px;">Certified ACS&#8805;99.7 w/w %</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Methanol</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>34860-4L-R</td>
			<td style="width:303px;">CHROMASOLV, for HPLC, &#8805;99.9%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Ammonium bicarbonate</td>
			<td style="width:155px;">Fluka</td>
			<td>09830-500G</td>
			<td style="width:303px;">BioUltra, &#8805;99.5% (T), Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Acetonitrile</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>34998-4L</td>
			<td style="width:303px;">CHROMASOLV Plus, for HPLC, &#8805;99.9%, Step 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Ethyl acetate</td>
			<td style="width:155px;">Fluka</td>
			<td>34972-1L-R</td>
			<td style="width:303px;">LC-MS CHROMASOLV, Step 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium borohydride</td>
			<td style="width:155px;">Aldrich</td>
			<td>213462-25G</td>
			<td style="width:303px;">ReagentPlus, 99%, Step 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Iodomethane</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>289566-100G</td>
			<td style="width:303px;">ReagentPlus, 99.5%,&#160; Step 6.&#160; Store at 4 &#176;C until use.&#160; Sit at room temperature before use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Sodium hydroxide solution, 50% w/w</td>
			<td style="width:155px;">Fisher</td>
			<td>SS254-1</td>
			<td style="width:303px;">Certified, Step 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dimethyl sulfoxide, anhydrous</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>276855-1L</td>
			<td style="width:303px;">Anhydrous, &#8805;99.9%, Step 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Methanol, anhydrous</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>322415-100ML</td>
			<td style="width:303px;">Anhydrous, 99.8%, Step 6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Dichloromethane</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>34856-4L</td>
			<td style="width:303px;">CHROMASOL&#174;, for HPLC, &#8805;99.8%, contains amylene as stabilizer, Step 7</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:231px;">Dowex 50WX8 hydrogen formhydrogen form 100-200&#160;mesh</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>217506-500G</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">AG 50W-X8 Resin&#160;</td>
			<td style="width:155px;">Bio-Rad</td>
			<td>142-1441</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:231px;">BAKERBOND spe 1 ml x 100 mg Solid Phase Extraction Column, PP, Octadecyl (C18) Reverse Phase</td>
			<td style="width:155px;">JT Baker</td>
			<td>JT-7020-01</td>
			<td style="width:303px;">Step 5 and 8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">7.5% Mini-PROTEAN TGX Precast Gel&#160;</td>
			<td style="width:155px;">Bio-Rad</td>
			<td></td>
			<td style="width:303px;">Step 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Bio-Safe Coomassie Stain&#160;</td>
			<td style="width:155px;">Bio-Rad</td>
			<td>161-0786</td>
			<td style="width:303px;">G-250, Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Silver Stain Kit for Mass Spectrometry</td>
			<td style="width:155px;">Pierce</td>
			<td>24600</td>
			<td style="width:303px;">Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Oligosaccharides Kit (Maltotriose, Dp3: R474140)</td>
			<td style="width:155px;">Supelco&#160;</td>
			<td>47265</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Oligosaccharides Kit (Maltotetraose, Dp4: R474135)</td>
			<td style="width:155px;">Supelco&#160;</td>
			<td>47265</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Disposable Pasteur Pipets, Glass, Short Tip</td>
			<td style="width:155px;">VWR</td>
			<td>14673-010</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">PYREX 13 x 100 mm Disposable Round Bottom Threaded Culture Tubes</td>
			<td style="width:155px;">Corning</td>
			<td>99447-13</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">PYREX 16 x 125 mm Disposable Round Bottom Threaded Culture Tubes</td>
			<td style="width:155px;">Corning</td>
			<td>99447-16</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Phenolic Caps/Closures with PTFE-Faced Rubber Liner</td>
			<td style="width:155px;">Kimble</td>
			<td>45066C-13</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Phenolic Caps/Closures with PTFE-Faced Rubber Liner</td>
			<td style="width:155px;">Kimble</td>
			<td>45066C-15</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>81265</td>
			<td style="width:303px;">volume 500&#160;&#956;L, needle size 22 ga&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>81165</td>
			<td style="width:303px;">volume 250&#160;&#956;L, needle size 22 ga</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>81065</td>
			<td style="width:303px;">volume 100&#160;&#956;L, needle size 22s ga&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>80965</td>
			<td style="width:303px;">volume 50&#160;&#956;L, needle size 22s ga</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton Calibrated Syringes</td>
			<td style="width:155px;">HAMILTON</td>
			<td>80300</td>
			<td style="width:303px;">volume 10&#160;&#956;L, needle size 26s ga&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">Petri Dish Glass 100mm x 15mm</td>
			<td style="width:155px;">GSC INTERNATIONAL INC</td>
			<td>1500-4</td>
			<td style="width:303px;">Wash before use, Step 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Bard-Parker Surgical Blades</td>
			<td style="width:155px;">Fisher</td>
			<td>371310</td>
			<td style="width:303px;">Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Reacti-Therm Heating/Stirring Module</td>
			<td style="width:155px;">Pierce</td>
			<td>18870</td>
			<td style="width:303px;">Step 1, 4 and 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Heating Blocks</td>
			<td style="width:155px;">Fisher</td>
			<td>125D</td>
			<td style="width:303px;">Step 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Pyrex fiber glass wool borosilicate pore size 8&#160;&#956;m</td>
			<td style="width:155px;">Aldrich</td>
			<td>CLS3950</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fused Silica CutterTubing cutter</td>
			<td style="width:155px;">alltech</td>
			<td>3194</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Multi-tube vortexers&#160;</td>
			<td style="width:155px;">VWR</td>
			<td>444-7063</td>
			<td style="width:303px;">Step 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Lyophilizer 25EL Freezemobile</td>
			<td style="width:155px;">Virtis</td>
			<td>25EL</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Centrifuge</td>
			<td style="width:155px;">VWR</td>
			<td>Clinical 50</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:231px;">LTQ Orbitrap Discovery</td>
			<td style="width:155px;">Thermo Fisher Scientific</td>
			<td></td>
			<td style="width:303px;">0</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="Fig...

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Improved In-gel Reductive &amp;#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.

質量分析法による総合的なO-結合およびスルホグライコミクスのための改良されたゲル内還元β脱離

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

Unit 1

Nucleophilic Substitution and Elimination Reactions of Alkyl Halides

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