Main content area

Facile synthesis of magnetic metal organic framework for highly efficient proteolytic digestion used in mass spectrometry-based proteomics

Zhai, Rui, Yuan, Yifeng, Jiao, Fenglong, Hao, Feiran, Fang, Xiang, Zhang, Yangjun, Qian, Xiaohong
Analytica chimica acta 2017
active sites, analytical chemistry, autolysis, chelating agents, coordination polymers, emus, immobilized enzymes, longevity, mass spectrometry, mice, oocytes, peptides, protein composition, proteins, proteolysis, proteome, proteomics, surface area, thermal stability, trypsin
Mass spectrometry (MS) based bottom-up strategy is now the first choice for proteomics analysis. In this process, highly efficient and complete enzymatic degradation of protein samples is extremely important to achieve in-depth protein coverage and high-throughput protein profiling. However, conventional in-solution digestion suffer from long digestion time and enzyme autolysis that limit the protein sample processing throughput and identification accuracy. Here, we developed a novel type of magnetic metal organic frameworks (MOFs)-based immobilized enzyme reactor, Fe3O4@DOTA-ZIF-90-trypsin. By introducing a stable chelator, 1,4,7,10-tetraazacyclododecane N,N′,N″,N‴-tetraacetic acid (DOTA), onto the magnetic cores, the hybrid supporting matrix of the immobilized enzyme reactor Fe3O4@DOTA-ZIF-90 has novel characteristics that include: i) favourable magnetic response (1.01 emu g−1) that makes the operation easy and convenient, ii) ultrahigh surface area (565.21 m2 g-11) and active sites that ensure high loading amounts and covalent linkage of enzyme, and iii) excellent structural and thermal stability that endows the immobilized enzyme reactor a prolonged lifespan. The performance of the magnetic MOFs-immobilized trypsin is first investigated using the standard protein, BSA, and the results showed that the immobilized enzyme reactor exhibits satisfactory digestion efficiency within only 1 min with the sequence coverage (80%) that is comparable or even better than that (70%) of the traditional 12 h-free trypsin digestion. To test the applicability of the magnetic MOFs-based immobilized enzyme reactor, protein samples extracted from 400 oocytes in mice were digested through the new immobilized enzyme reactor. In total, 8957 peptides corresponding to 1843 protein groups are identified, which are nearly of 40% and 67% increases in the number of identified proteins and peptides compared to using in-solution digestion with free proteases. Specifically, the identification of oocyte-specific proteins was critical in the discovery of and understanding the regulation mechanism of oocyte maturation. Thus, this synthetic procedure of Fe3O4@DOTA-ZIF-90 provides a universal method for fabrication of magnetic MOFs materials, and the successful application of Fe3O4@DOTA-ZIF-90-trypsin in efficient protein digestion for deeper proteome coverage will undoubtedly enlarge the uses for MOFs.