Department of Measurement Technology and Industrial Electrical Engineering, Division of Nanobiotechnology, Lund University, Box 118, SE-211 00 Lund, Sweden.
Anal Chem. 2012 Oct 16;84(20):8663-9. doi: 10.1021/ac3017983. Epub 2012 Sep 24.
A major bottleneck in high-throughput protein production is the validation step, which is why parallel and automated sample processing methods are highly desirable. Also, a miniaturized sample preparation format is preferred, as the reduction of reagent volumes significantly decreases the analysis cost per sample. We have developed an automated and miniaturized protein sequence verification protocol for recombinant proteins utilizing peptide mass fingerprinting and MS/MS analysis. The integrated selective enrichment target (ISET) platform, previously developed in our group, with its dual functionality, being both a sample preparation platform and a MALDI target plate, is employed. All steps including immobilized metal ion affinity chromatography of protein on cobalt-loaded beads, tryptic digestion, and MALDI MS analysis are performed in an array format, without any sample transfers, on the same ISET chip. The automated configuration reduced the sample preparation time significantly. Starting with crude lysate, a full plate of 48 purified, digested samples prepared for MALDI-MS can be generated in 4 h, with only 30 min of operator involvement. This paper demonstrates the utility of the method by parallel analysis of 45 His-tagged human recombinant proteins.
高通量蛋白质生产的一个主要瓶颈是验证步骤，这就是为什么需要并行和自动化的样品处理方法。此外，还需要采用微型化的样品制备格式，因为减少试剂体积可以显著降低每个样品的分析成本。我们已经开发了一种利用肽质量指纹图谱和 MS/MS 分析的重组蛋白的自动化和微型化蛋白质序列验证协议。该协议利用了我们小组之前开发的集成选择性富集靶标（ISET）平台，该平台具有双重功能，既是样品制备平台，也是 MALDI 靶板。所有步骤，包括钴负载珠上蛋白质的固定化金属离子亲和层析、胰蛋白酶消化和 MALDI MS 分析，都在同一 ISET 芯片上以阵列格式进行，无需任何样品转移。自动化配置大大缩短了样品制备时间。从粗裂解物开始，仅需 30 分钟的操作人员参与，即可在 4 小时内生成一整板 48 个纯化、消化的用于 MALDI-MS 的样品。本文通过平行分析 45 个人类重组 His 标签蛋白证明了该方法的实用性。
