Quantitative Characterization of Glycopeptide Isomers
糖肽异构体的定量表征
基本信息
- 批准号:10540152
- 负责人:
- 金额:$ 34.22万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2019
- 资助国家:美国
- 起止时间:2019-02-01 至 2027-01-31
- 项目状态:未结题
- 来源:
- 关键词:2019-nCoVAddressAdhesionsAldehydesAlgorithmsAlzheimer&aposs DiseaseAmidesAminationAmino AcidsAmmoniumAreaBacterial AdhesinsBiologicalBiological ProcessBlood capillariesCOVID-19CalibrationCarbonCationsCell CommunicationCell Culture TechniquesCell LineCellsChargeChromatographyCommunitiesComputer softwareComputing MethodologiesCystic FibrosisDataDegenerative polyarthritisDementiaDerivation procedureDevelopmentDiabetes MellitusDiagnosticDigestionDiseaseDisease ProgressionDrug TargetingGlucoseGlutamineGlycopeptidesGlycoproteinsGoalsHydrophobicityImmune responseIn VitroIndividualInvestigationIsomerismLabelLaboratoriesLinkLiquid ChromatographyMalignant NeoplasmsMammalian CellMediatingMetabolicMethodsMonitorOutcomePeptidesPolysaccharidesPost-Translational Protein ProcessingProcessProtein GlycosylationProteinsReactionReagentReportingReproducibilityResearchResolutionSamplingSialic AcidsSialoglycopeptidesSideSodiumSoftware ToolsStable Isotope LabelingStructureTechnologyTimeVertebral columnVirus DiseasesWorkanalytical methodanalytical toolbiological systemscomputerized data processingdrug developmentglycoproteomicsglycosylationimprovedindexinginnovationinstrumentinterestionizationmetaperiodatenovelopen sourceoxidationpathogensialylationtandem mass spectrometry
项目摘要
Because of the pressing needs to comprehensively understand the biological attributes of glycosylation in
many critical biological functions such as the immune response, cell development, cellular
differentiation/adhesion and host-pathogen interactions, glycoproteomics continues to be a highly dynamic
research area. Aberrant glycosylation for decades has been recognized as the attribute of many mammalian
diseases, including osteoarthritis, cystic fibrosis, and cancer. Moreover, isomeric alterations of glycoproteins
have been observed in diseases such as Alzheimer’s Disease and cancers. Recently, glycans and their isomers
have been reported to be vital to the SARS-CoV-2 viral infection, making them a crucial target for the drug
development of COVID-19. Therefore, reliable, and efficient characterization of glycopeptides and their isomers
is necessary to better understand the attributes of glycosylation in biological and biomedical processes. We are
proposing here four specific aims: Aim 1. To enhance the separation and identification of glycopeptide isomers
using mesoporous graphitized carbon (MGC)-LC-MS and hydrophobicity index of peptides (HIP); Aim 2. To
enhance the quantification of glycopeptide isomers using a 15N metabolic-TMT multiplexing approach and a
parallel reaction monitoring (PRM) method; Aim 3. To enhance the glycopeptide isomeric characterization using
novel derivatization methods; and Aim 4. To enhance automated isomeric glycopeptide data processing by the
development of improved software. The outcome of these aims will provide reliable and efficient glycoproteomic
platforms and algorithms for a better isomeric characterization of glycopeptides which can be employed to
address biomedical issue, thus contributing to the glycoscience community. The innovations of this proposal
originate from the uniqueness of the proposed analytical methods and software. The isomeric separation of
glycopeptides using MGC-LC-MS/MS is a highly innovative method, developed in our lab, permitting efficient
separation of glycopeptide isomers on a 1 cm short column. The retention time normalization of glycans intra
and inter-laboratories has been introduced and demonstrated to be necessary previously through a glucose unit
index (GUI), but not in glycoproteomic analysis. For the first time, we will investigate the retention time
normalization of glycopeptides and glycopeptide isomers using a set of peptides which have known
hydrophobicity factors on different instruments and different laboratories. The combination of 15N stable isotope
labeling of glycopeptides and TMT will double the multiplexing capacity of TMT to 36-plex when studying in vitro
cell line glycoproteomics. Although PRM has been utilized for glycoproteomic profiling, the analysis of isomeric
glycoproteomics is lacking. Thus, this will be the first comprehensive investigation of glycopeptide isomers using
PRM. Moreover, the derivatization methods we proposed are of great innovation. It will be the first time to achieve
2-aminobenzamide (2-AB) labeling on sialic acids of glycopeptides, and the first time to achieve efficient isomeric
separation of sialoglycopeptides on a 15 cm C18 column via a two-step oxidation-reductive amination reaction.
In addition, we have achieved a derivatization of sialylated glycopeptides (DOSG) method that will introduce
mass difference to distinguish α2,3 and α2,6 linked sialoglycopeptides. Consequently, the combination oxidation-
reductive amination and DOSG methods leads to an innovative enrichment method for sialoglycopeptides. The
derivatization of carboxyl groups of sialic acids on glycans and amino acids on peptide backbone neutralizes
additional charges and the addition of quaternary ammonium functionalized molecules provides controllable
positive charges which make the enrichment using strong cation exchange (SCX) possible. This is a novel
method for the efficient enrichment of sialoglycopeptides where sialic acid linkage isomers can be distinguished
at the same time. The deliverables of this proposal are reliable, adaptable, and affordable strategies and
improved software to enhance the isomeric glycopeptide studies by any laboratory interested in defining
comprehensive protein glycosylation using LC-MS/MS. The proposed technologies are expected to enable a
better understanding of the biological attributes of glycoprotein isomers in the development and progression of
diseases.
由于迫切需要全面了解糖基化的生物学属性,
许多关键的生物学功能,如免疫反应、细胞发育、细胞免疫、细胞免疫和细胞免疫。
尽管糖蛋白质组学在分化/粘附和宿主-病原体相互作用方面具有重要的作用,但糖蛋白质组学仍然是一个高度动态的研究领域。
研究领域。几十年来,异常糖基化已被认为是许多哺乳动物的属性,
疾病,包括骨关节炎、囊性纤维化和癌症。此外,糖蛋白的异构改变
在阿尔茨海默氏症和癌症等疾病中观察到。最近,聚糖及其异构体
据报道,它们对SARS-CoV-2病毒感染至关重要,这使它们成为该药物的重要靶点
COVID-19的发展。因此,糖肽及其异构体的可靠和有效表征
是必要的,以更好地了解生物和生物医学过程中的糖基化的属性。我们
在此提出四个具体目标:目标1。加强糖肽异构体的分离鉴定
使用介孔石墨化碳(MGC)-LC-MS和肽的疏水性指数(HIP);目的2.到
使用15 N代谢-TMT多重方法和
平行反应监测(PRM)方法;目的3。为了增强糖肽异构体表征,
新的衍生化方法;和目的4。为了增强自动化异构糖肽数据处理,
开发更好的软件。这些目标的结果将提供可靠和有效的糖蛋白质组学
用于更好地表征糖肽异构体的平台和算法,
解决生物医学问题,从而为糖科学界做出贡献。该提案的创新之处在于
这源于所提出的分析方法和软件的独特性。异构体分离
糖肽的MGC-LC-MS/MS是我们实验室开发的一种高度创新的方法,
在1cm短柱上分离糖肽异构体。聚糖的保留时间归一化
和实验室间已经引入,并证明是必要的,以前通过葡萄糖单位
索引(GUI),但不是在糖蛋白质组学分析。我们将首次调查保留时间
使用已知的一组肽对糖肽和糖肽异构体进行标准化
不同仪器和不同实验室的疏水因子。15 N稳定同位素
在体外研究时,糖肽和TMT的标记将使TMT的多重能力加倍至36重
细胞系糖蛋白组学尽管PRM已被用于糖蛋白质组学分析,但异构体的分析仍然是一个难题。
缺乏糖蛋白组学。因此,这将是第一次全面调查的糖肽异构体使用
PRM.此外,我们提出的衍生化方法具有很大的创新性。这将是第一次实现
2-氨基苯甲酰胺(2-AB)标记糖肽的唾液酸,并首次实现了高效异构化,
通过两步氧化-还原胺化反应在15 cm C18柱上分离唾液酸糖肽。
此外,我们还实现了唾液酸化糖肽衍生化(DOSG)方法,
质量差异以区分α 2,3和α 2,6连接的唾液酸糖肽。因此,组合氧化-
还原胺化和DOSG方法导致了唾液酸糖肽的创新富集方法。的
聚糖上唾液酸的羧基和肽骨架上氨基酸的衍生化
另外的电荷和季铵官能化分子的加入提供了可控的
正电荷,这使得使用强阳离子交换(SCX)进行富集成为可能。这是一部小说
可区分唾液酸键异构体的有效富集唾液酸糖肽的方法
同时还研究与讨论该提案的交付成果是可靠、适应性强和负担得起的战略,
改进的软件,以增强任何有兴趣定义的实验室对糖肽异构体的研究
使用LC-MS/MS进行全面的蛋白质糖基化。
更好地了解糖蛋白异构体的生物学属性,
疾病
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Yehia Mechref其他文献
Yehia Mechref的其他文献
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{{ truncateString('Yehia Mechref', 18)}}的其他基金
Quantitative Characterization of Glycopeptide Isomers
糖肽异构体的定量表征
- 批准号:
10331873 - 财政年份:2019
- 资助金额:
$ 34.22万 - 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
- 批准号:
8787914 - 财政年份:2014
- 资助金额:
$ 34.22万 - 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
- 批准号:
10019565 - 财政年份:2014
- 资助金额:
$ 34.22万 - 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
- 批准号:
8927045 - 财政年份:2014
- 资助金额:
$ 34.22万 - 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
- 批准号:
10318016 - 财政年份:2014
- 资助金额:
$ 34.22万 - 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
- 批准号:
10697345 - 财政年份:2014
- 资助金额:
$ 34.22万 - 项目类别:
Sensitive and Quantitative MS-bases Glycomic Mapping Platform
基于 MS 的灵敏定量糖组图谱平台
- 批准号:
9120382 - 财政年份:2014
- 资助金额:
$ 34.22万 - 项目类别:
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