This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.
Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides (“glycan nodes”) present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.
糖脂,糖蛋白,蛋白聚糖,和葡糖胺聚糖构成统称为聚糖复杂的异构碳水化合物的四个主要类。如质膜,多糖包被和细胞外基质和流体的普遍存在和积分分量,聚糖参加等不同生化过程如细胞内吞作用,细胞内运输,细胞运动性,信号转导,分子识别,受体活化,细胞粘附,宿主 – 病原体相互作用,细胞间通讯,免疫监视和免疫反应的启动。1几乎存在于各个领域的生活,被称为糖基转移酶的建立聚糖的聚合物的酶与糖苷水解酶(又称糖苷酶,它分解聚糖)建造,改造串联行动,最终产生敲定聚糖聚合物2。虽然每个糖基转移酶可以在不同的糖结合物进行操作,糖基转移酶通常由特定激活核苷酸的糖供体( 例如,GDP-岩藻糖)的单糖部分转移到亲核受体的某一类别( 例如,脂质,多肽,核酸,或生长伪造linkage-和端基异构体特异性糖苷键低聚糖)。据估计,蛋白质(尤其是膜和分泌蛋白)的50%以上是在翻译后糖基化修饰的3-初步组合计算提供了相当大的变异性,多功能性和特异性给予通过糖基化的糖蛋白的欣赏。例如,如果一个多肽基片具有仅10的糖基化位点和每个站点可以与只有3种不同的单糖还原末端的1糖苷键,那么,从理论上说,最后的糖蛋白可以假定3 10 = 59049不同身份。在糖蛋白糖苷键通常与侧链氮Ø形成序列的Asn-X-丝氨酸f中的天冬酰胺残基/苏氨酸(X可以是除脯氨酸外的任何氨基酸),得到N- -glycans 2和侧链羟基丝氨酸和苏氨酸残基,以得到õ-glycans 4。一个细胞的糖组的组成(即它的糖基化产物补码)是唯一的,有限的,因为,除了少数例外,糖基转移酶表现出严格的供体,受体和联动特异性。5重要和丰富的血浆糖蛋白遭受异常的糖基化作为下游的后果异常糖基转移酶表达和活性的由于许多病理情况下,尤其是癌症和炎性疾病。6-24
主要是由于后生因素的影响,糖组是显著更加多样化,动态和复杂的比蛋白质组和转录组25,26虽然哺乳动物基因组的约1%的编码形成,修改和聚糖的组装,在一个非模板驱动方式-一个明显的对比27的糖基化进行到多肽和核酸的生物合成。糖基化酶和诸如环境因素如营养物和前体的可用性的相对数量和活性之间的相互影响,最终决定的性质,速率,和糖基化的程度。5,28胚胎发生( 例如,测定和分化),细胞活化,和进展通过细胞周期的影响的基因表达( 即,转录和翻译),并改变提供糖基转移酶的特性和量,其活性是细胞的聚糖谱的直接上游的决定因素。因为(部分的)的增殖,粘合剂,和癌细胞的侵入性质类似于那些普通胚发生细胞中,聚糖的生物合成途径( 例如,前体积累,失调的表达,aberran具体变化的吨修改,结构截短,或新颖的形成)作为普遍的癌症生物标志物指示的肿瘤形成,进展,迁移和侵袭29各个阶段虽然糖基化是高度复杂的,显然仅在糖基化几改变可以使发生和转移。显然,某些“异常”糖基化产物的确使他们逃避免疫识别和生存在恶劣的血管和转移性环境迁移的需求,有利于癌细胞。28,30,31毫不奇怪,实验表明,破坏或阻止的模式改变基因表达和异常聚糖形成可阻止肿瘤的发生。29尽管如此,生物流体样本( 例如,尿液,唾液,和血浆或血清)中检测出的异常聚糖可能不是癌症直接指标(或其它疾病),而是下游的微妙而显著成果在免疫系统或以不可预知的器官有害条件量化后果变化。32
虽然它们提供了关于糖组通用的信息,许多分子基于交互的糖组学技术( 例如,外源凝集素/抗 体阵列和代谢/共价标记)取决于检测整个聚糖结构,并且不提供关于单个聚糖的详细结构信息。形成鲜明对比,质谱(MS)可以帮助识别和量化个别聚糖结构并揭示这种结构的信息作为连接位点到多肽芯。失调的表达或仅一个糖基转移酶的活性可以发起在多个糖基化途径有害分子事件的级联。因为每一个糖基转移酶可能不止一个糖缀合物基板上,并在不同的成长多糖聚合物运作,放松管制的生物合成级联产生disproportionally增加只有一个聚糖产品的量,但几个异构类细胞内或细胞外液异常聚糖。33,然而,这些独特的异常聚糖有时被认为是不切实际的作为生物标志物用于癌症或其它聚糖-情感病症,因为,相对于大池的良好调节聚糖,这些异常聚糖代表一小部分是可能甚至常常由这种高度敏感的技术如质谱仍然检测不到。例如,在细胞内和细胞外体液,广泛蛋白质浓度谱(跨越8个数量级)可以防止由该更丰富的物种掩蔽稀少的糖蛋白的检测。32此外,在确定糖基转移酶活仍然相当实用和理论的挑战,因为许多糖基转移酶在临床生物液体缺席或处于非活动状态体外 。尽管consisten难度TLY检测和定量独具特色的聚糖的超微量,质谱的从业者对用人完好聚糖作为临床指标取得了巨大进步。我们最近开发了一种互补的方法来完整聚糖的分析是,采用GC-MS,便于对所有构成分支点和特定键的单糖(“聚糖节点”),它们一起赋予唯一每个聚糖并且在许多的检测案件直接服务的量化有罪糖基转移酶(S)的相对活性分子的代理人。
自从其首次报道于1958年的直接应用到聚糖分析,气相色谱(GC)已经证明一个功能强大的技术来分析每甲基化的单-和二糖,34确定其anomericity和绝对构型,并用于随后的质谱分析将它们分开。35从1984年到2007年,Ciucanu和他的同事引入并精制2005至2008年间所采用氢氧化钠和碘甲烷使用水和氯仿。35,36固相聚糖全甲基技术,接着全甲基聚糖的液/液萃取,康和同事集成了省时的旋-column方式进入全甲基一步。37,38在2008年,戈茨研究小组设计了一个定量的固相全甲基聚糖分析方法采用基质辅助激光解吸电离(MALDI)质谱分析比较和区分潜在的侵入性和非-invasive乳腺癌细胞;然后39,2009年,戈茨团队结合酶和化学释放技术40切割Ø-glycans从完整的糖蛋白在强碱性固相全甲基方案虽然格茨程序同时促进和全甲基化学释放。 Ø-glycans的,它仅适用于预分离的糖蛋白质。我们在2013年修改的这种技术和通过掺入三氟乙酸适于它为整个未分级的生物流体和均质化的组织样本(TFA)水解,还原和乙酰化的步骤。33,这些额外的步骤也释放从糖脂和N-聚糖从糖蛋白-连接的聚糖,并转换他们进入部分甲基化的糖醇乙酸酯(PMAAs, 图1),其独特的甲基化和乙酰化的图案便于通过GC-MS和唯一地分析表征原始完整聚糖聚合物41( 图2)中的构成聚糖节点33最终,这过程产生在复杂的生物流体的所有聚糖的复合肖像基于独特的聚糖的功能,如“核心岩藻糖基”,“6唾液酸化”,“平分GlcNAc的”,和“beta 1-6支链”直接的,相对定量 – 每个从单个GC-MS chromatograp衍生HIC高峰。本文介绍的全甲基的进一步优化,乙酰化,分离,并用在相对定量的方式改进沿着清理阶段。
在一般情况下,成功地生产部分甲基化的糖醇乙酸酯的(PMAAs)从己糖是充满了很多困难,并且比成功制造N- -acetylhexosamine(HexNAc)PMAAs的更稳健。后面,因为它发挥出在这个过程中的每一步该现象的确切机制是未知的,但必须与本-N-乙酰基(而不是羟基)的独特的化学是唯一相对于己糖HexNAcs。后面,因为它涉及酸水解该现象的机制在别处。43说明总之,对于N -methylaceta…
The authors have nothing to disclose.
This work was supported by the College of Liberal Arts and Sciences of Arizona State University in the form of laboratory startup funds to CRB. It was also supported by a grant from Flinn Foundation (Grant No. 1977) and by the National Cancer Institute of the National Institutes of Health under Award Number R33CA191110. JA was supported by the National Institute of General Medical Sciences of the National Institutes of Health Postbaccalalureate Research Education Program (PREP) under award number R25GM071798. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Sodium hydroxide beads | 367176 | Sigma-Aldrich | 20-40 mesh, reagent grade 97% |
0.9 mL Spin column | 69705 | Pierce division of ThermoFisher Scientific | Includes plugs and polyethylene frits |
GC-MS autosampler vial (silanized)* | C4000-9 | ThermoFisher Scientific | Target DP High Recovery Vial, 1.5 mL, 12 x 32 mm, Includes Teflon-lined pierceable caps |
1.5 mL polypropylene test tubes | 05-402-25 | ThermoFisher Scientific | Snap-cap lid |
2 mL polypropylene test tubes | 05-408-138 | ThermoFisher Scientific | Snap-cap lid |
Dimethyl Sulfoxide (DMSO) | D8418 | Sigma-Aldrich | BioReagent for molecular biology, reagent grade >99.0% |
Iodomethane | I8507 | Sigma-Aldrich | Contains copper as stabilizer, ReagentPlus 99% |
Acetonitrile | A955-4 | ThermoFisher Scientific | Optima LC/MS |
Microcentrifuge | 75002436: Sorvall Legend Micro 17 Centrifuge | ThermoFisher Scientific | 24 x 1.5/2.0 rotor with ClickSeal biocontainment lid. Rotor catalog number: 75003424 |
13 x 100 glass test tube (silanized)* | 53283-800 | VWR | 13 x 100 mm borosilicate glass test tubes with screw-cap finish |
Caps for glass test tubes | 14-930-15D | ThermoFisher Scientific | Kimble™ Black Phenolic Screw Caps; 13mm-415 GPI thread; PTFE-faced rubber liner. |
Sodium chloride | S7653 | Sigma-Aldrich | >99.5% pure |
Chloroform | 4440-08 | Macron Fine Chemicals | |
Trifluoroacetic acid | 299537 | Sigma-Aldrich | 99% purified by redistillation for protein sequencing |
Sodium borohydride | 71321 | Fluka Analytical | 99% |
Ammonium hydroxide solution | 320145 | Sigma-Aldrich | NH3 content: 28.0 – 30.0% |
Methanol | AH230-4 | Honeywell Burdick & Jackson | HPLC grade |
Acetic acid | 320099 | Sigma-Aldrich | 99.70% |
Plastic vacuum desiccator | Any model of adequate size | FoodSaver | |
Acetic anhydride | 539996 | Sigma-Aldrich | 99.50% |
Dichloromethane | D143SK-4 | ThermoFisher Scientific | Stabilized HPLC grade |
Acetone | 9006-03 | J.T.Baker | Baker Analyzed |
Heated evaporation manifold (main unit) | pi18823 | ThermoFisher Scientific | Thermo Scientific* Reacti-Therm* Heating and Stirring Module; Triple-block Model with Heating and Stirring Function |
Heated evaporation manifold (overhead evaporator) | pi18826 | ThermoFisher Scientific | ThermoScientific* Reacti-Vap Evaporator, 27-port; For use with triple-block Reacti-Therm heating module |
Aluminum sample-holder blocks for evaporation manifold | pi18816 | ThermoFisher Scientific | Block, Aluminum, Reacti-Block S-1, Holds 13mm dia test tubes, 13 holes (14 dia. x 45mm deep) |
Gas chromatograph | Model A7890 | Agilent | Equipped with CTC PAL autosampler |
Mass spectrometer | GCT Premier (Time-of-Flight) | Waters | |
Split-mode liner (deactivated / silanized) | 5183-4647 | Agilent | Containing a small plug of silanized glass wool |
DB-5ms GC column | 122-5532 | Agilent | 30 m x 0.25 mm ID x 0.25 micron film thickness |
Chlorotrimethylsilane | 95541 | Sigma-Aldrich | |
Glass vacuum desiccator (for glassware silanization) | EW-06536-30 | Cole-Parmer | 12" wide; 230 mm plate size |
*Glassware silanization is carried out in-house, overnight using chlorotrimethylsilane vapor in a large glass vacuum desiccator. |