AN LC/MS PLATFORM FOR REDUCTIVE AMINATION-TAGGED GLYCANS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 通过还原胺化化学，使用稳定同位素聚糖标签，有助于质谱聚糖定量。 这样的标签可以与释放的含有醛还原末端的聚糖一起使用。 在MSR开发了一种四链体标签，并使用N-聚糖和糖胺聚糖进行了测试。 两种聚糖类别均可使用酰胺-亲水相互作用色谱法（HILIC），使用质谱兼容的移动的相进行分离。 为了对聚糖表达进行定量比较，有必要开发一个平台，该平台允许稳健和可重复的数据采集以及方便的数据简化。 作为这项工作的一部分，MSR正在与安捷伦科技公司合作，测试基于芯片的聚糖LC/MS喷雾接口。芯片喷雾器装有酰胺-二氧化硅固定相，并使用N-聚糖、肝素、硫酸乙酰肝素和硫酸软骨素进行测试。 结果表明，对于所有测试的聚糖类别，可以容易地获得非常稳健和稳定的喷雾。 聚糖倾向于在初始结果中产生样品残留。 这个问题应该可以通过更广泛地清洗自动进样器针头来解决。 数据由质量色谱图组成，其中在2-3个电荷状态下观察到给定聚糖组成。 最低电荷状态通常在铵加合物中观察到。 因此，每种组合物观察为4-5 m/z值。 典型的混合物由几十种成分组成，产生非常复杂的光谱模式。 此次合作的目的是开发用于提取、去卷积和合并所有LC/MS离子的软件程序，以便将数据简化为易于解释的聚糖组成和丰度表。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mass spectrometric glycan quantitation is facilitated by the use of stable isotope glycan tags through reductive amination chemistry. Such tags may be used with released glycans containing an aldehyde reducing end. A tetraplex tag has been developed at the MSR and tested using N-glycans and glycosaminoglycans. Both glycan classes may be separated using amide-hydrophilic interaction chromatography (HILIC) using mass spectrometry compatible mobile phases. In order to make quantitative comparisons of glycan expression, it is necessary to develop a platform that allows robust and reproducible data acquisition and facile data reduction. As part of this effort, the MSR is collaborating with Agilent Technologies Corp. to test a chip-based spray interface for glycan LC/MS. The chip sprayer was packed with amide-silica stationary phase and tested using N-glycans, heparin, heparan sulfates, and chondroitin sulfates. The results showed that very robust and stable spray could be easily obtained for all glycan classes tested. The glycans tended to produce sample carry over in initial results. This problem should be solvable with more extensive washing of the autosampler needle. The data consist of mass chromatograms in which a given glycan composition is observed in 2-3 charge states. The lowest charge state is typically observed with ammonium adducts. Thus, each composition is observed as 4-5 m/z values. A typical mixture consistes of several dozen compositions, giving rise to very complex spectral patterns. It is objective of this collaboration to develop software routines for extracting, de-convoluting and combining all LC/MS ions so as to reduce the data into readily interpretable glycan composition and abundance tables.