Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres To Achieve Automated Proteome Quantification

Protein digestion and isotope labeling are two critical steps in proteome quantification. However, the conventional in-solution protocol unavoidably suffers from disadvantages such as time-consuming, low labeling efficiency, and tedious off-line manual operation, which might affect the quantification accuracy, reproducibility, and throughput. To address these problems, we developed a fully automated proteome quantification platform, in which an ultraperformance immobilized microreactor (upIMER) with graphene-oxide-modified polymer microspheres as the matrix was developed, to achieve not only the simultaneous protein digestion and ¹⁸O labeling, but also the online integration with nano-high-pressure liquid chromatography–electrospray ionization-tandem mass spectrometry (nanoHPLC–ESI-MS/MS). Compared to the conventional off-line protocols, such a platform exhibits obviously improved digestion and ¹⁸O labeling efficiency (only 8% peptides with missed cleavage sites, 99% labeling efficiency, and 2.5 min reaction time), leading to the increased quantification coverage, accuracy, precision and throughput. All the results demonstrated that our developed fully automated platform should provide new opportunities to improve the accuracy, reproducibility, and throughput for proteome quantification.