Wang Yonghui, Wu Shiaw-Lin, Hancock William S
Barnett Institute, Northeastern University, 360 Huntington Ave., Boston, Massachusetts 02115, USA.
Biotechnol Prog. 2006 May-Jun;22(3):873-80. doi: 10.1021/bp060005l.
In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.
在本文中，我们描述了将凝集素色谱与毛细管液相色谱联用，并与线性离子阱-傅里叶变换质谱仪（LTQ/FTMS）相结合，以从细胞培养裂解物中富集和鉴定过表达的糖蛋白。一种特征明确的糖蛋白，重组组织型纤溶酶原激活剂（rt-PA），被用作标准品，并且我们证明了rt-PA标准品胰蛋白酶消化产物中存在的三种N-连接糖肽（包括聚糖结构）可以在新的混合质谱平台上进行鉴定。该方法的一个特点是，通过直接分析胰蛋白酶消化产物，无需额外耗时的样品制备方案，就可以获得大量关于碳水化合物结构的信息。然后研究了凝集素的组合，以提高捕获糖肽的回收率，并将其与不同凝集素对特定糖基化基序的选择性相关联。然后将该方法扩展到生物技术生产中常用的细胞系（中国仓鼠卵巢细胞，CHO）的裂解物。这项研究表明，凝集素的组合可以从CHO细胞裂解物中显著富集糖蛋白。我们还证明，通过这种富集水平和新的混合质谱仪，我们可以通过在FTMS中进行精确质量测量以及在线性离子阱中进行串联MSn，以1:200（rtPA:总细胞裂解物蛋白，w/w）的比例研究粗CHO细胞裂解物中存在的rt-PA的N-连接糖肽的结构。凝集素方法的通用性和高通量性质，以及直接分析胰蛋白酶消化产物中聚糖结构的能力，表明该平台有潜力在生物技术产业的早期生产阶段常规监测糖蛋白产品。
