改善凝胶还原β-消除综合O连接和磺基糖组学质谱
Summary
In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive β-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.
Abstract
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 β-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.
Introduction
糖基化是一个重要的蛋白质翻译后修饰，有助于有机体生理学，组织病理学和细胞识别1-3。尽管在分析glycoscience重大进展，在一个特定的蛋白聚糖特征的完整的多样性仍然是一个极具挑战性的任务，特别是对蛋白质的主要生物来源分离出来。然而，糖蛋白的聚糖的微观不均经常会影响与其它蛋白质功能性相互作用。因此，聚糖多样性的表征对于理解细胞和组织中的糖基化4,5-生理意义是必不可少的。为了了解糖蛋白的糖基化对组织的生理与病理生理学，健壮，灵敏，全面的glycomic分析技术的贡献已经变得越来越重要。在蛋白质组分析，蛋白质的鉴定是通过LC-MS通常实现/胰蛋白酶肽6的MS分析。蛋白消化可使用以下在凝胶内消化以蛋白酶如胰蛋白酶7-9纯化的蛋白或蛋白通过SDS-PAGE解析来进行。预富集通过SDS-PAGE的蛋白质混合物的增强的蛋白质的ID的深度和准确性。类似的战略糖蛋白的糖基化glycomic分析的发展，关键在glycoscience的前列。
聚糖的两类主要是通过是N-连接或O-键附着到蛋白质骨架。 N-连接的聚糖附着到天冬酰胺（天冬酰胺）中所定义的的Asn-X-丝氨酸/苏氨酸/半胱氨酸一序列子的部分残基（X为任意氨基酸除脯氨酸外的），并且可以通过酶消化与肽N为释放-glycanase（PNGaseF或A），无论是在溶液中，在-凝胶法，或对印迹10-12。 O-连接的糖链主要附着到丝氨酸（丝氨酸）或苏氨酸（Thr）残基。但是，只有一个酶已经IDENtified，其能够从糖蛋白释放的O-联聚糖的，它有一个极其有限聚糖特异性，只释放最简单的O-连接的聚糖。化学释放策略仍首选从糖蛋白全面释放的O-连接的糖链的方法。还原性或非还原性β-消除，或肼是良好表征的化学释放技术和目前最常用的方法为从糖蛋白13,14释放O-连接的聚糖。虽然还原性β-消除已被用于从糖蛋白，通过SDS-PAGE分离释放O-连接的聚糖，先前的方法需要HPLC分离用于随后的分析15-17。
多维质谱（MS n）的分析，目前提供的数据结构为特征的糖蛋白中分离出大多数生物源预计的数额发布聚糖最丰富的来源。 MS的深度基于结构特征是由permethylating释放聚糖之前，他们的分析大大便利。全甲基增强离子化，容易在广泛的聚糖结构18,19的均衡摩尔信号的响应。此外，全甲基明确标记终端和取代的单糖部分具有鲜明的群众，从而提高结构解析20-23。例如，酸性聚糖一般难以检测作为非全甲基化物种被MS。虽然酸性聚糖可以在负离子模式通过质谱进行检测，就不可能检测酸性和中性聚糖在同一离子模式。聚糖全甲基的一个主要优点是，所有的聚糖的单糖取代基的游离羟基（OH）的将甲基（OCH 3或OMe），因此唾液酸化聚糖的电荷被中和被封端的，这使得它们作为可检测如全甲基化中性（亚洲LO）聚糖。然而，硫酸盐部分的上sulfoglycans的羟基是抗全甲基，导致保留阴离子电荷，从而抑制离子化和降低灵敏度。这种抑制目前防止的非常复杂的糖蛋白，如粘蛋白，它携带高丰硫酸化多糖24-26综合glycomic分析。
上纯化的硫酸化聚糖的最近的报道用过充电，反相层析，纯化和之前的MALDI分析单独的全甲基化的聚糖。此方法依赖于使用用于洗脱不同的流动相，这是我们发现比有机相分配不那么严格的硫酸化和非硫酸化聚糖的完全分离。因此，适合于sulfoglycans的检测和富集新技术是这里提出。这些技术允许的硫酸化聚糖的以下水的定量回收在水相中：DCM（二氯甲烷）提取，这是经常在聚糖全甲基反应27结束时执行。重要的是，从全 甲基非硫酸化多糖的混合物，这种强大的分离全甲基sulfoglycans的伴随丰富的带电物质，同时也简化了MS 2碎裂模式。一个全面的协议，用于改善胶O连接聚糖分析也提出了。改进协议增强聚糖恢复，增加了获得的通过级质谱全甲基化聚糖的分析的结构信息，并提高了施加到从生物来源分离的必不可少的糖蛋白sulfoglycomic分析的灵敏度。
此协议旨在用于通过SDS-PAGE分离感兴趣的特定糖蛋白的全提取物的糖蛋白O-连接的糖链的分析或与由三个实验程序; A）凝胶清理，B）的凝胶还原β-淘汰，C）聚糖permethy分页。我们的目标是获得全面的O连接glycomic数据糖蛋白生物的兴趣（ 图1）的主要来源收获。糖蛋白通过SDS-PAGE分离是通过染色和感兴趣的条带切下，将所得的凝胶带被切成小块的可视化。将凝胶片被脱色，并进行乙酸乙酯洗涤以除去凝胶污染物（ 图2A）。聚糖释放是通过在凝胶还原性β-消除（ 图2B）来实现与所释放的糖链进行全甲基化。水-有机萃取下面的全甲基定量分割阴离子硫酸化聚糖远离非硫酸化的中性聚糖（ 图2C）。在凝胶还原β-消除耦合到水 – 有机萃取使的O-连接的聚糖，以及少量的糖蛋白，通过SDS-PAGE分离的释放sulfoglycans表征。该战略概述研究概述zed有在图1中，其细节示于图2中 。此外，可用于通过标准的LC-MS / MS的蛋白质组技术蛋白的ID的脱色和洗涤凝胶片的一部分上。
Protocol
Representative Results
Discussion
Combining in-gel reductive β-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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Water, deionized water | Sigma-Aldrich | 270733-4L | CHROMASOLV, for HPLC |
| Acetic acid, Glacial | Fisher | A38-500 | Certified ACS≥99.7 w/w % |
| Methanol | Sigma-Aldrich | 34860-4L-R | CHROMASOLV, for HPLC, ≥99.9% |
| Ammonium bicarbonate | Fluka | 09830-500G | BioUltra, ≥99.5% (T), Step 1 |
| Acetonitrile | Sigma-Aldrich | 34998-4L | CHROMASOLV Plus, for HPLC, ≥99.9%, Step 1 |
| Ethyl acetate | Fluka | 34972-1L-R | LC-MS CHROMASOLV, Step 1 |
| Sodium borohydride | Aldrich | 213462-25G | ReagentPlus, 99%, Step 2 |
| Iodomethane | Sigma-Aldrich | 289566-100G | ReagentPlus, 99.5%, Step 6. Store at 4 °C until use. Sit at room temperature before use. |
| Sodium hydroxide solution, 50% w/w | Fisher | SS254-1 | Certified, Step 6 |
| Dimethyl sulfoxide, anhydrous | Sigma-Aldrich | 276855-1L | Anhydrous, ≥99.9%, Step 6 |
| Methanol, anhydrous | Sigma-Aldrich | 322415-100ML | Anhydrous, 99.8%, Step 6 |
| Dichloromethane | Sigma-Aldrich | 34856-4L | CHROMASOL®, for HPLC, ≥99.8%, contains amylene as stabilizer, Step 7 |
| Dowex 50WX8 hydrogen formhydrogen form 100-200 mesh | Sigma-Aldrich | 217506-500G | Step 3 |
| AG 50W-X8 Resin | Bio-Rad | 142-1441 | Step 3 |
| BAKERBOND spe 1 ml x 100 mg Solid Phase Extraction Column, PP, Octadecyl (C18) Reverse Phase | JT Baker | JT-7020-01 | Step 5 and 8 |
| 7.5% Mini-PROTEAN TGX Precast Gel | Bio-Rad | Step 1 | |
| Bio-Safe Coomassie Stain | Bio-Rad | 161-0786 | G-250, Step 1 |
| Silver Stain Kit for Mass Spectrometry | Pierce | 24600 | Step 1 |
| Oligosaccharides Kit (Maltotriose, Dp3: R474140) | Supelco | 47265 | |
| Oligosaccharides Kit (Maltotetraose, Dp4: R474135) | Supelco | 47265 | |
| Disposable Pasteur Pipets, Glass, Short Tip | VWR | 14673-010 | Wash before use |
| PYREX 13 x 100 mm Disposable Round Bottom Threaded Culture Tubes | Corning | 99447-13 | Wash before use |
| PYREX 16 x 125 mm Disposable Round Bottom Threaded Culture Tubes | Corning | 99447-16 | Wash before use |
| Phenolic Caps/Closures with PTFE-Faced Rubber Liner | Kimble | 45066C-13 | Wash before use |
| Phenolic Caps/Closures with PTFE-Faced Rubber Liner | Kimble | 45066C-15 | Wash before use |
| Hamilton HPLC syringe | HAMILTON | 81265 | volume 500 μL, needle size 22 ga |
| Hamilton HPLC syringe | HAMILTON | 81165 | volume 250 μL, needle size 22 ga |
| Hamilton HPLC syringe | HAMILTON | 81065 | volume 100 μL, needle size 22s ga |
| Hamilton HPLC syringe | HAMILTON | 80965 | volume 50 μL, needle size 22s ga |
| Hamilton Calibrated Syringes | HAMILTON | 80300 | volume 10 μL, needle size 26s ga |
| Petri Dish Glass 100mm x 15mm | GSC INTERNATIONAL INC | 1500-4 | Wash before use, Step 1 |
| Bard-Parker Surgical Blades | Fisher | 371310 | Step 1 |
| Reacti-Therm Heating/Stirring Module | Pierce | 18870 | Step 1, 4 and 7 |
| Heating Blocks | Fisher | 125D | Step 2 |
| Pyrex fiber glass wool borosilicate pore size 8 μm | Aldrich | CLS3950 | Step 3 |
| Fused Silica CutterTubing cutter | alltech | 3194 | Step 3 |
| Multi-tube vortexers | VWR | 444-7063 | Step 6 |
| Lyophilizer 25EL Freezemobile | Virtis | 25EL | |
| Centrifuge | VWR | Clinical 50 | |
| LTQ Orbitrap Discovery | Thermo Fisher Scientific | 0 |
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