School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia.
Rapid Commun Mass Spectrom. 2014 Mar 15;28(5):471-82. doi: 10.1002/rcm.6806.
Diseases including cancer and congenital disorders of glycosylation have been associated with changes in the site-specific extent of protein glycosylation. Saliva can be non-invasively sampled and is rich in glycoproteins, giving it the potential to be a useful biofluid for the discovery and detection of disease biomarkers associated with changes in glycosylation.
Saliva was collected from healthy individuals and glycoproteins were enriched using phenylboronic acid based glycoprotein enrichment resin. Proteins were deglycosylated with peptide-N-glycosidase F and digested with AspN or trypsin. Desalted peptides and deglycosylated peptides were separated by reversed-phase liquid chromatography and detected with on-line electrospray ionization quadrupole-time-of-flight mass spectrometry using a 5600 TripleTof instrument. Site-specific glycosylation occupancy was semi-quantitatively determined from the abundance of deglycosylated and nonglycosylated versions of each given peptide.
Glycoprotein enrichment identified 67 independent glycosylation sites from 24 unique proteins, a 3.9-fold increase in the number of glycosylation sites identified. Enrichment of glycoproteins rather than glycopeptides allowed detection of both deglycosylated and nonglycosylated versions of each peptide, and thereby robust measurement of site-specific occupancy at 21 asparagines. Healthy individuals showed limited biological variability in occupancy, with partially modified sites having characteristics consistent with inefficient glycosylation by oligosaccharyltransferase. Inclusion of negative controls without enzymatic deglycosylation controlled for spontaneous chemical deamidation, and identified asparagines previously incorrectly annotated as glycosylated.
We developed a sample preparation and mass spectrometry detection strategy for rapid and efficient measurement of site-specific glycosylation occupancy on diverse salivary glycoproteins suitable for biomarker discovery and detection of changes in glycosylation occupancy in human disease.
包括癌症和先天性糖基化紊乱在内的疾病与蛋白质糖基化的特定部位的程度变化有关。唾液可以非侵入性地取样，并且富含糖蛋白，使其有可能成为用于发现和检测与糖基化变化相关的疾病生物标志物的有用生物流体。
从健康个体中收集唾液，并使用基于苯硼酸的糖蛋白富集树脂富集糖蛋白。使用肽-N-糖苷酶 F 将蛋白质去糖基化，并使用 AspN 或胰蛋白酶进行消化。用反相液相色谱分离脱糖肽和去糖基化肽，并使用在线电喷雾电离四极杆飞行时间质谱仪（5600 TripleTof 仪器）进行检测。通过对每个给定肽的去糖基化和非糖基化版本的丰度进行半定量测定，确定了特定部位的糖基化占有率。
糖蛋白富集从 24 个独特蛋白质中鉴定出 67 个独立的糖基化位点，糖基化位点的鉴定数量增加了 3.9 倍。糖蛋白的富集而不是糖肽的富集允许检测到每个肽的去糖基化和非糖基化版本，从而可以对 21 个天冬酰胺的特定部位占有率进行稳健测量。健康个体的占有率显示出有限的生物学变异性，部分修饰的位点具有寡糖基转移酶催化效率低下的糖基化特征。不进行酶去糖基化的阴性对照的纳入控制了自发的化学脱酰胺作用，并鉴定了以前错误注释为糖基化的天冬酰胺。
我们开发了一种样品制备和质谱检测策略，用于快速有效地测量各种唾液糖蛋白上特定部位的糖基化占有率，适合用于生物标志物的发现和检测人类疾病中糖基化占有率的变化。
