Zhu Zhikai, Go Eden P, Desaire Heather
The Ralph N. Adams Institute for Bioanalytical Chemistry and Department of Chemistry, University of Kansas, 2030 Becker Drive, Lawrence, KS, 66047, USA.
J Am Soc Mass Spectrom. 2014 Jun;25(6):1012-7. doi: 10.1007/s13361-014-0859-2. Epub 2014 Mar 27.
N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.
N-连接聚糖是维持蛋白质适当生物学功能所必需的。糖基化不足会导致动植物出现多种疾病；因此，表征蛋白质上的糖基化程度是理解、诊断和治疗疾病的重要一步。为了确定糖基化位点占有率，通常使用蛋白质N-糖苷酶F（PNGase F）从蛋白质上分离聚糖，在此过程中，先前糖基化的天冬酰胺会脱酰胺变成天冬氨酸。通过比较所得含天冬氨酸的肽与含非糖基化天冬酰胺的肽的丰度，可以评估糖基化位点占有率。然而，当非糖基化天冬酰胺发生自发化学脱酰胺时，这种方法可能会给出不准确的结果。为了克服这一局限性，我们开发了一种新的测量糖基化位点占有率的方法，该方法不依赖于将糖基化肽转化为去糖基化形式。具体而言，通过使用重同位素标记的内标结合液相色谱-质谱分析，同时对蛋白质的总浓度和非糖基化部分进行定量，并准确确定位点占有率。通过对牛胎球蛋白上一个糖基化位点的占有率进行定量，证明了该方法的有效性。所开发的方法是第一项不使用PNGase F测量糖基化位点占有率的工作，并且由于在整个工作流程中聚糖保持完整，因此可以与糖肽分析并行进行。
