Search

Rationale: Thermal proteome profiling (TPP) has been widely used for the identification of drug targets for several years, and TMTpro‐16plex has recently been evaluated for TPP of vehicle‐ and drug‐treated samples in a single labeling process to reduce missing values and save instrument time. A novel isobaric labeling reagent, IBT‐16plex, was developed with slightly better performance in protein identification and quantification than the commercially available TMTpro‐16plex. Methods: In this study, we applied the newly developed IBT‐16plex for target identification of methotrexate and panobinostat using TPP. Results: The known targets of these two drugs were successfully identified with elevated melting temperatures, and some known off‐targets and potential new off‐targets were also identified. Conclusions: IBT‐16plex can be a cost‐effective replacement for TMTpro‐16plex for TPP applications. [ABSTRACT FROM AUTHOR]

Protein glycosylation in eukaryotic cells regulates a variety of physiological processes including cell recognition, cell adhesion, migration, and immune response. It is also closely related with the occurrence and development of many critical diseases. Therefore, large scale identification of protein glycosylation not only provides important information for the study of basic biological mechanisms, but also is crucial for the discovery of new diagnostic biomarkers and therapeutic targets. Due to the low abundance of glycoprotein/glycopeptide in real biological samples, enrichment before mass spectrometry (MS) analysis is an essential step for achieving deep glycosylation site coverage. Lectin enrichment, as an effective method for glycoproteins/glycopeptides enrichment, has been utilized widely in glycoproteomics research. To solve the problems of low lectin loading and limited enrichment efficiency of existing lectin functional materials, we prepared two kinds of new graphene oxide ( GO) immobilized lectin. Besides good dispersion in aqueous solution as well as good chemical stability, GO has extremely large specific surface area and also carries high density of functional groups on its surface, which is especially benelicial for achieving high lectin loading amount. As a result, lectin loading as high as 1. 90 mg/mg was achieved for GO-lectin (GO-ConA 2. 073 mg/mg, RSD = 1. 0%; GO-WGA 1. 908 mg/mg, RSD = 0.14%). One milligram GO-lectin can adsorb more than 200 µg glycoprotein each experiment in two weeks. The GO-lectin was successfully applied in glycoproteins/glycopeptides enrichment with high efficiency and selectivity, indicating its good application potential in glycoproteomics research.