TOX4 is one of the regulatory factors of PP1 phosphatases with poorly understood functions. Here we show that chromatin occupancy pattern of TOX4 resembles that of RNA polymerase II (Pol II), and its loss increases cellular level of C-terminal domain (CTD) phosphorylated Pol II but mainly decreases Pol II occupancy on promoters. In addition, elongation rate analyses by 4sUDRB-seq suggest that TOX4 restricts pause release and early elongation but promotes late elongation. Moreover, TT-seq analyses indicate that TOX4 loss mainly decreases transcriptional output. Mechanistically, TOX4 may restrict pause release through facilitating CTD serine 2 and DSIF dephosphorylation, and promote Pol II recycling and reinitiation through facilitating CTD serines 2 and 5 dephosphorylation. Furthermore, among the PP1 phosphatases, TOX4 preferentially binds PP1α and is capable of facilitating Pol II CTD dephosphorylation in vitro. These results lay the foundation for a better understanding of the role of TOX4 in transcriptional regulation.

Isobaric labeling is the most widely used multiplexing quantitative approach in proteomic studies, enabling the comparison of up to 18 samples in a single MS analysis. Expanding the multiplexing capacity is of great necessity for high-throughput proteomic studies. Herein, we establish a novel TAG-TMTpro approach by introducing Ala or Gly residues to peptides prior to TMTpro labeling, which is able to triple the quantitative capacity of TMTpro. We systematically evaluated the Boc-Ala-OSu and Boc-Gly-OSu reaction and optimized the conditions for labeling, side-product elimination, and Boc deprotection. We validated the identification and quantification performance using E. coli and HeLa cell lysates. We demonstrated that the TAG-TMTpro approach resulted in good identification reproducibility and reliable quantitative accuracy. The TAG-TMTpro is able to triple the multiplexing capacity of TMTpro reagents and is a versatile quantitative approach for high-throughput proteomic studies. Data are available via ProteomeXchange with identifier PXD033711.

Isobaric labeling has emerged as an indispensable quantitative proteomic approach for its unprecedented multiplexing capacity in a single analysis. Currently, different hyperplexing approaches have been developed to meet the demand for the increasing sample size in large-scale cohort analysis. In this report, we present a tribrid hyperplexing approach by the combinatorial use of three types of isobaric reagents, a novel isobaric tag 16-plex (IBT16) reagent and the widely used tandem mass tag (TMT; TMT11) and TMTpro (TMT18) reagents. After the determination of labeling efficiency and the optimization of testing conditions, we systematically evaluated the identification and quantification performance of the three labeling reagents in both independent and combinatorial manners using the mixtures of E. coli and HeLa peptides with different ratios. Our results reveal that the three reagents are quite similar in all testing aspects despite some differences, and the combination use of the three reagents could expand the multiplexing capacity to up to 45-plex. Furthermore, we conclude the advantages of IBT16 in the combination use and the preferred combinations for different practical applications. Data are available via ProteomeXchange with identifier PXD037498.