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.