Department of Analytical Chemistry, Ruhr-University Bochum, Germany.
J Proteomics. 2011 Apr 1;74(4):502-9. doi: 10.1016/j.jprot.2011.01.005. Epub 2011 Jan 26.
The enrichment of glycosylated proteins by glycocapturing materials plays a pivotal role for the investigation of polysaccharide containing proteins in disease pathogenesis. Hence, we investigated a boric acid gel as a binding material for glycoprotein enrichment. The bovine proteins alpha-1-acid-glycoprotein (A1AG) and alpha-2-HS-glycoprotein (fetuin A) were spiked in human chronic wound fluids and were subsequently enriched by a boric acid gel affinity chromatography (BAGAC). The enrichment efficiency was evaluated by western blot analysis and mass spectrometry. Additionally, glycoproteins of human wound fluids from diabetes mellitus patients with chronic foot ulcers were analyzed after BAGAC enrichments. In total 104 glycoproteins were identified, with reported glycosylation sites. 60 proteins were detected in at least 2 out of 3 biological replicates and were used for quantitative analysis between the bound and unbound fractions. Almost 80% of these glycoproteins were more prominent in the bound fraction. Only 2 glycoproteins revealed higher spectral counts in the flow through fraction compared to the bound fraction. These findings demonstrate the capability of the BAGAC material to enrich glycosylated proteins from complex human wound fluids.
糖基化蛋白的糖捕获材料富集在研究多糖结合蛋白在疾病发病机制中的作用至关重要。因此，我们研究了硼酸凝胶作为糖蛋白富集的结合材料。将牛蛋白α-1-酸性糖蛋白（A1AG）和α-2-HS-糖蛋白（胎球蛋白 A）掺入人慢性伤口液中，然后通过硼酸凝胶亲和层析（BAGAC）进行富集。通过 Western blot 分析和质谱法评估富集效率。此外，还分析了糖尿病慢性足部溃疡患者人伤口液经 BAGAC 富集后的糖蛋白。总共鉴定出 104 种糖蛋白，其中报道了糖基化位点。在至少 3 个生物学重复中的 2 个中检测到 60 种蛋白质，并在结合和未结合部分之间进行定量分析。这些糖蛋白中近 80%在结合部分更为突出。与结合部分相比，只有 2 种糖蛋白在流动通过部分的光谱计数更高。这些发现证明了 BAGAC 材料从复杂的人伤口液中富集糖基化蛋白的能力。
