Analysis of Protein Glycosylation after Rapid Digestion Using Protease-Containing Membranes in Spin Columns
Analysis of Protein Glycosylation after Rapid Digestion Using Protease-Containing Membranes in Spin Columns
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DOI：
10.1021/jasms.3c00038
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发表时间：
2023-05-01
影响因子：
3.2
通讯作者：
Bruening，Merlin L.
中科院分区：
文献类型：
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作者：
Cao，Weikai;Bruening，Merlin L.
糖基化是一种重要的蛋白质翻译后修饰，其在治疗性蛋白质的生物活性和病毒蛋白质的感染性中起关键作用。液相色谱-串联质谱法很容易鉴定具有位点特异性的蛋白聚糖。然而，在常规的溶液中蛋白水解中使用的过夜孵育导致糖基化分析的高周转时间，特别是当位点特异性聚糖鉴定需要连续的溶液中双链酶时。使用牛胎球蛋白作为模型糖蛋白，这项工作首先表明，与过夜的溶液中消化相比，在103分钟内的膜内消化产生类似的聚糖鉴定和定量。在旋转柱中的含蛋白酶的膜能够在100 - 30秒内消化治疗性蛋白质（曲妥珠单抗和促红细胞生成素）和病毒蛋白质（SARS-CoV-2受体结合域）。溶液内和膜内消化后的聚糖鉴别相似，有限的膜内消化增强了曲妥珠单抗中高甘露糖聚糖的鉴别。最后，含有胰蛋白酶和胰凝乳蛋白酶的堆叠膜允许快速连续的蛋白水解消化以位点特异性地鉴定SARS-CoV-2受体结合结构域的聚糖。人们可以很容易地在商业旋转柱中组装含蛋白酶的膜，同时旋转多个柱将有助于并行分析。
Glycosylation is an important protein post-translational modification that plays a pivotal role in the bioactivity of therapeutic proteins and in the infectivity of viral proteins. Liquid chromatography with tandem mass spectrometry readily identifies protein glycans with site specificity. However, the overnight incubation used in conventional in-solution proteolysis leads to high turnaround times for glycosylation analysis, particularly when sequential in-solution digestions are needed for site-specific glycan identification. Using bovine fetuin as a model glycoprotein, this work first shows that in-membrane digestion in ∼3 min yields similar glycan identification and quantitation when compared to overnight in-solution digestion. Protease-containing membranes in a spin column enable digestion of therapeutic proteins (trastuzumab and erythropoietin) and a viral protein (SARS-CoV-2 receptor binding domain) in ∼30 s. Glycan identification is similar after in-solution and in-membrane digestion, and limited in-membrane digestion enhances the identification of high-mannose glycans in trastuzumab. Finally, stacked membranes containing trypsin and chymotrypsin allow fast sequential proteolytic digestion to site-specifically identify the glycans of SARS-CoV-2 receptor binding domain. One can easily assemble the protease-containing membranes in commercial spin columns, and spinning multiple columns simultaneously will facilitate parallel analyses.