To elucidate the relationship between the protein function and the diversity and heterogeneity of glycans conjugated to the protein, glycosylation sites, glycan variation, and glycan proportions at each site of the glycoprotein must be analyzed. Glycopeptide-based structural analysis technology using mass spectrometry has been developed; however, complicated analyses of complex spectra obtained by multistage fragmentation are necessary, and sensitivity and throughput of the analyses are low. Therefore, we developed a liquid chromatography/mass spectrometry (MS)-based glycopeptide analysis method to reveal the site-specific glycome (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile, Glyco-RIDGE). This method used accurate masses and retention times of glycopeptides, without requiring MS2, and could be applied to complex mixtures. To increase the number of identified peptide, fractionation of sample glycopeptides for reduction of sample complexity is required. Therefore, in this study, glycopeptides were fractionated into four fractions by hydrophilic interaction chromatography, and each fraction was analyzed using the Glyco-RIDGE method. As a result, many glycopeptides having long glycans were enriched in the highest hydrophilic fraction. Based on the monosaccharide composition, these glycans were thought to be poly-N-acetyllactosamine (polylactosamine [pLN]), and 31 pLN-carrier proteins were identified in HL-60 cells. Gene ontology enrichment analysis revealed that pLN carriers included many molecules related to signal transduction, receptors, and cell adhesion. Thus, these findings provided important insights into the analysis of the glycoproteome using our novel Glyco-RIDGE method.

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为了阐明蛋白质功能与偶联到蛋白质上的聚糖的多样性和异质性之间的关系，必须分析糖蛋白每个位点的糖基化位点，聚糖变异和聚糖比例。已经开发了使用质谱技术的基于糖肽的结构分析技术；然而，通过多级裂解获得的复杂光谱的复杂分析是必要的，并且分析的灵敏度和通量较低。因此，我们开发了一种基于液相色谱/质谱（MS）的糖肽分析方法，以揭示位点特异性糖基（洗脱谱上糖肽信号的基于糖类异质性的关系识别），Glyco-RIDGE。此方法使用了糖肽的精确质量和保留时间，而无需使用MS2，并可以应用于复杂的混合物。为了增加鉴定出的肽的数量，需要对样品糖肽进行分级分离以降低样品的复杂性。因此，在本研究中，通过亲水相互作用色谱将糖肽分为四个级分，并使用Glyco-RIDGE方法对每个级分进行了分析。结果，许多具有长聚糖的糖肽富含最高的亲水级分。基于单糖的组成，这些聚糖被认为是聚 并使用Glyco-RIDGE方法分析每个级分。结果，许多具有长聚糖的糖肽富含最高的亲水性部分。基于单糖的组成，这些聚糖被认为是聚 并使用Glyco-RIDGE方法分析每个级分。结果，许多具有长聚糖的糖肽富含最高的亲水性部分。基于单糖的组成，这些聚糖被认为是聚在HL-60细胞中鉴定出N-乙酰基乳糖胺（polylactosamine [pLN]）和31种pLN载体蛋白。基因本体论富集分析表明，pLN载体包括许多与信号转导，受体和细胞粘附有关的分子。因此，这些发现为使用我们新颖的Glyco-RIDGE方法分析糖蛋白组提供了重要的见识。

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