Abstract：
As glycosylations are difficult to analyze, their roles and effects are poorly understood. Glycosylations in human milk (HM) differ across lactation. Glycosylations can be involved in antimicrobial activities and may serve as food for beneficial microorganisms. This study aimed to identify and analyze O-linked glycans in HM by high-throughput mass spectrometry. 184 longitudinal HM samples from 66 donors from day 3 and months 1, 2, and 3 postpartum were subjected to a post-translational modification specific enrichment-based strategy using TiO2 and ZrO2 beads for O-linked glycopeptide enrichment. β-CN was found to be a major O-linked glycoprotein, additionally, αS1-CN, κ-CN, lactotransferrin, and albumin also contained O-linked glycans. As glycosyltransferases and glycosidases are involved in assembling the glycans including O-linked glycosylations, these were further investigated. Some glycosyltransferases and glycosidases were found to be significantly decreasing through lactation, including two O-linked glycan initiator enzymes (GLNT1 and GLNT2). Despite their decrease, the overall level of O-linked glycans remained stable in HM over lactation. Three different motifs for O-linked glycosylation were enriched in HM proteins: Gly-Xxx-Xxx-Gly-Ser/Thr, Arg-Ser/Thr and Lys-Ser/Thr. Further O-linked glycan motifs on β-CN were observed to differ between intact proteins and endogenous peptides in HM.
摘要：
由于糖基化难以分析，人们对它们的作用和影响知之甚少。人乳(HM)中的糖基化在整个泌乳期不同。糖基化可以参与抗微生物活性并且可以作为有益微生物的食物。本研究旨在通过高通量质谱鉴定和分析HM中的O-连接聚糖。对来自产后第3天和第1、2和3个月的66个供体的184个纵向HM样品进行翻译后修饰特异性富集策略，使用TiO2和ZrO2珠进行O-连接的糖肽富集。β-CN被发现是一种主要的O-连接糖蛋白，此外，αS1-CN,κ-CN,乳转铁蛋白,和白蛋白还含有O-连接的聚糖。由于糖基转移酶和糖苷酶参与组装聚糖，包括O-连接的糖基化，这些被进一步调查。发现一些糖基转移酶和糖苷酶在哺乳期显着减少，包括两种O-连接的聚糖引发酶(GLNT1和GLNT2)。尽管减少了,泌乳期HM中O-连接聚糖的总体水平保持稳定.HM蛋白中富集了三种不同的O连接糖基化基序：Gly-Xxx-Xxx-Gly-Ser/Thr，Arg-Ser/Thr和LysSer/Thr.观察到β-CN上的其他O-连接的聚糖基序在HM中的完整蛋白质和内源性肽之间不同。
