TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples.

Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs-all with essentially no missing values across the 16 samples and no loss in quantitative integrity.

Architecture of the human interactome defines protein communities and disease networks.

The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.

Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis.

NCOA4 is a selective cargo receptor for the autophagic turnover of ferritin, a process critical for regulation of intracellular iron bioavailability. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting.

A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors.

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins.