Dopaminergic neurons modulate neural circuits and behaviors via dopamine (DA) release from expansive, long range axonal projections. The elaborate cytoarchitecture of these neurons is embedded within complex brain tissue, making it difficult to access the neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell-type specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of midbrain dopaminergic neurons. Our dataset reveals the proteomic architecture underlying proteostasis, axonal metabolism, and neurotransmission in these neurons. We find that most proteins encoded by DA neuron-enriched genes are localized within striatal dopaminergic axons, including ion channels with previously undescribed axonal localization. These proteomic datasets provide a resource for neuronal cell biology, and this approach can be readily adapted for study of other neural cell types.

The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.

Esophageal cancer (EC) is a fatal digestive disease with a poor prognosis and frequent lymphatic metastases. Nevertheless, reliable biomarkers for EC diagnosis are currently unavailable. Accordingly, we have performed a comparative proteomics analysis on cancer and paracancer tissue-derived exosomes from eight pairs of EC patients using label-free quantification proteomics profiling and have analyzed the differentially expressed proteins through bioinformatics. Furthermore, nano-flow cytometry (NanoFCM) was used to validate the candidate proteins from plasma-derived exosomes in 122 EC patients. Of the 803 differentially expressed proteins discovered in cancer and paracancer tissue-derived exosomes, 686 were up-regulated and 117 were down-regulated. Intercellular adhesion molecule-1 (CD54) was identified as an up-regulated candidate for further investigation, and its high expression in cancer tissues of EC patients was validated using immunohistochemistry, real-time quantitative PCR (RT-qPCR), and western blot analyses. In addition, plasma-derived exosome NanoFCM data from 122 EC patients concurred with our proteomic analysis. The receiver operating characteristic (ROC) analysis demonstrated that the AUC, sensitivity, and specificity values for CD54 were 0.702, 66.13%, and 71.31%, respectively, for EC diagnosis. Small interference (si)RNA was employed to silence the CD54 gene in EC cells. A series of assays, including cell counting kit-8, adhesion, wound healing, and Matrigel invasion, were performed to investigate EC viability, adhesive, migratory, and invasive abilities, respectively. The results showed that CD54 promoted EC proliferation, migration, and invasion. Collectively, tissue-derived exosomal proteomics strongly demonstrates that CD54 is a promising biomarker for EC diagnosis and a key molecule for EC development.

Hearing and balance rely on small sensory hair cells that reside in the inner ear. To explore dynamic changes in the abundant proteins present in differentiating hair cells, we used nanoliter-scale shotgun mass spectrometry of single cells, each ~1 picoliter, from utricles of embryonic day 15 chickens. We identified unique constellations of proteins or protein groups from presumptive hair cells and from progenitor cells. The single-cell proteomes enabled the de novo reconstruction of a developmental trajectory using protein expression levels, revealing proteins that greatly increased in expression during differentiation of hair cells (e.g., OCM, CRABP1, GPX2, AK1, GSTO1) and those that decreased during differentiation (e.g., TMSB4X, AGR3). Complementary single-cell transcriptome profiling showed corresponding changes in mRNA during maturation of hair cells. Single-cell proteomics data thus can be mined to reveal features of cellular development that may be missed with transcriptomics.

The tubular network is a critical part of the endoplasmic reticulum (ER). The network is shaped by the reticulons and REEPs/Yop1p that generate tubules by inducing high membrane curvature, and the dynamin-like GTPases atlastin and Sey1p/RHD3 that connect tubules via membrane fusion. However, the specific functions of this ER domain are not clear. Here, we isolated tubule-based microsomes from Saccharomyces cerevisiae via classical cell fractionation and detergent-free immunoprecipitation of Flag-tagged Yop1p, which specifically localizes to ER tubules. In quantitative comparisons of tubule-derived and total microsomes, we identified a total of 79 proteins that were enriched in the ER tubules, including known proteins that organize the tubular ER network. Functional categorization of the list of proteins revealed that the tubular ER network may be involved in membrane trafficking, lipid metabolism, organelle contact, and stress sensing. We propose that affinity isolation coupled with quantitative proteomics is a useful tool for investigating ER functions.

Improvements in LC-MS/MS methods and technology have enabled the identification of thousands of modified peptides in a single experiment. However, protein regulation by post-translational modifications (PTMs) is not binary, making methods to quantify the modification extent crucial to understanding the role of PTMs. Here, we introduce FLEXIQuant-LF, a software tool for large-scale identification of differentially modified peptides and quantification of their modification extent without knowledge of the types of modifications involved. We developed FLEXIQuant-LF using label-free quantification of unmodified peptides and robust linear regression to quantify the modification extent of peptides. As proof of concept, we applied FLEXIQuant-LF to data-independent-acquisition (DIA) data of the anaphase promoting complex/cyclosome (APC/C) during mitosis. The unbiased FLEXIQuant-LF approach to assess the modification extent in quantitative proteomics data provides a better understanding of the function and regulation of PTMs. The software is available at https://github.com/SteenOmicsLab/FLEXIQuantLF.

CRM1 is a highly conserved, RanGTPase-driven exportin that carries proteins and RNPs from the nucleus to the cytoplasm. We now explored the cargo-spectrum of CRM1 in depth and identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈1000 from Xenopus oocytes and >1050 from human cells. In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes. The data suggest new CRM1 functions in spatial control of vesicle coat-assembly, centrosomes, autophagy, peroxisome biogenesis, cytoskeleton, ribosome maturation, translation, mRNA degradation, and more generally in precluding a potentially detrimental action of cytoplasmic pathways within the nuclear interior. There are also numerous new instances where CRM1 appears to act in regulatory circuits. Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction.

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+, and Ca2+, and thus shapes cellular excitability, plasticity, and metabolic activity. The molecular appearance and operation of TRPM7 channels in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative mass spectrometry (MS) analysis. We found that native TRPM7 channels are high-molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ADP-ribosylation factor-like protein 15 (ARL15). Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that complex formation effectively and specifically impacts TRPM7 activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

Vesicular neurotransmitter transporters (VNTs) mediate the selective uptake and enrichment of small-molecule neurotransmitters into synaptic vesicles (SVs) and are therefore a major determinant of the synaptic output of specific neurons. To identify novel VNTs expressed on SVs (thus identifying new neurotransmitters and/or neuromodulators), we conducted localization profiling of 361 solute carrier (SLC) transporters tagging with a fluorescent protein in neurons, which revealed 40 possible candidates through comparison with a known SV marker. We parallelly performed proteomics analysis of immunoisolated SVs and identified seven transporters in overlap. Ultrastructural analysis further supported that one of the transporters, SLC35D3, localized to SVs. Finally, by combining metabolite profiling with a radiolabeled substrate transport assay, we identified UDP-glucose as the principal substrate for SLC35D3. These results provide new insights into the functional role of SLC transporters in neurotransmission and improve our understanding of the molecular diversity of chemical transmitters.

Posttranslational modifications (PTMs) play a crucial role in a wide range of biological processes. Lysine crotonylation (Kcr) is a newly discovered histone PTM that is enriched at active gene promoters and potential enhancers in mammalian cell genomes. However, the cellular enzymes that regulate the addition and removal of Kcr are unknown, which has hindered further investigation of its cellular functions. Here we used a chemical proteomics approach to comprehensively profile 'eraser' enzymes that recognize a lysine-4 crotonylated histone H3 (H3K4Cr) mark. We found that Sirt1, Sirt2, and Sirt3 can catalyze the hydrolysis of lysine crotonylated histone peptides and proteins. More importantly, Sirt3 functions as a decrotonylase to regulate histone Kcr dynamics and gene transcription in living cells. This discovery not only opens opportunities for examining the physiological significance of histone Kcr, but also helps to unravel the unknown cellular mechanisms controlled by Sirt3, that have previously been considered solely as a deacetylase.

The role of pro-inflammatory macrophage activation in cardiovascular disease (CVD) is a complex one amenable to network approaches. While an indispensible tool for elucidating the molecular underpinnings of complex diseases including CVD, the interactome is limited in its utility as it is not specific to any cell type, experimental condition or disease state. We introduced context-specificity to the interactome by combining it with co-abundance networks derived from unbiased proteomics measurements from activated macrophage-like cells. Each macrophage phenotype contributed to certain regions of the interactome. Using a network proximity-based prioritization method on the combined network, we predicted potential regulators of macrophage activation. Prediction performance significantly increased with the addition of co-abundance edges, and the prioritized candidates captured inflammation, immunity and CVD signatures. Integrating the novel network topology with transcriptomics and proteomics revealed top candidate drivers of inflammation. In vitro loss-of-function experiments demonstrated the regulatory role of these proteins in pro-inflammatory signaling.

A decline of skeletal muscle strength with aging is a primary cause of mobility loss and frailty in older persons, but the molecular mechanisms of such decline are not understood. Here, we performed quantitative proteomic analysis from skeletal muscle collected from 58 healthy persons aged 20 to 87 years. In muscle from older persons, ribosomal proteins and proteins related to energetic metabolism, including those related to the TCA cycle, mitochondria respiration, and glycolysis, were underrepresented, while proteins implicated in innate and adaptive immunity, proteostasis, and alternative splicing were overrepresented. Consistent with reports in animal models, older human muscle was characterized by deranged energetic metabolism, a pro-inflammatory environment and increased proteolysis. Changes in alternative splicing with aging were confirmed by RNA-seq analysis. We propose that changes in the splicing machinery enables muscle cells to respond to a rise in damage with aging.

The spatiotemporal proteome of the intervertebral disc (IVD) underpins its integrity and function. We present DIPPER, a deep and comprehensive IVD proteomic resource comprising 94 genome-wide profiles from 17 individuals. To begin with, protein modules defining key directional trends spanning the lateral and anteroposterior axes were derived from high-resolution spatial proteomes of intact young cadaveric lumbar IVDs. They revealed novel region-specific profiles of regulatory activities and displayed potential paths of deconstruction in the level- and location-matched aged cadaveric discs. Machine learning methods predicted a 'hydration matrisome' that connects extracellular matrix with MRI intensity. Importantly, the static proteome used as point-references can be integrated with dynamic proteome (SILAC/degradome) and transcriptome data from multiple clinical samples, enhancing robustness and clinical relevance. The data, findings, and methodology, available on a web interface (http://www.sbms.hku.hk/dclab/DIPPER/), will be valuable references in the field of IVD biology and proteomic analytics.

Malaria is caused by infection of the erythrocytes by the parasites Plasmodium. Inside the erythrocytes, the parasites multiply via schizogony, an unconventional cell division mode. The inner membrane complex (IMC), an organelle located beneath the parasite plasma membrane, serving as the platform for protein anchorage, is essential for schizogony. So far, the complete repertoire of IMC proteins and their localization determinants remain unclear. Here we used biotin ligase (TurboID)-based proximity labeling to compile the proteome of the schizont IMC of the rodent malaria parasite Plasmodium yoelii. In total, 300 TurboID-interacting proteins were identified. 18 of 21 selected candidates were confirmed to localize in the IMC, indicating good reliability. In light of the existing palmitome of Plasmodium falciparum, 83 proteins of the P. yoelii IMC proteome are potentially palmitoylated. We further identified DHHC2 as the major resident palmitoyl-acyl-transferase of the IMC. Depletion of DHHC2 led to defective schizont segmentation and growth arrest both in vitro and in vivo. DHHC2 was found to palmitoylate two critical IMC proteins CDPK1 and GAP45 for their IMC localization. In summary, this study reports an inventory of new IMC proteins and demonstrates a central role of DHHC2 in governing the IMC localization of proteins during the schizont development.

Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn's disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.

Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed; however, proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae.

The combined use of multiple omics allows to study complex interrelated biological processes in their entirety. We applied a combination of metabolomics, lipidomics and proteomics to human bones to investigate their combined potential to estimate time elapsed since death (i.e., the postmortem interval [PMI]). This 'ForensOMICS' approach has the potential to improve accuracy and precision of PMI estimation of skeletonized human remains, thereby helping forensic investigators to establish the timeline of events surrounding death. Anterior midshaft tibial bone was collected from four female body donors before their placement at the Forensic Anthropology Research Facility owned by the Forensic Anthropological Center at Texas State (FACTS). Bone samples were again collected at selected PMIs (219-790-834-872days). Liquid chromatography mass spectrometry (LC-MS) was used to obtain untargeted metabolomic, lipidomic, and proteomic profiles from the pre- and post-placement bone samples. The three omics blocks were investigated independently by univariate and multivariate analyses, followed by Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies (DIABLO), to identify the reduced number of markers describing postmortem changes and discriminating the individuals based on their PMI. The resulting model showed that pre-placement metabolome, lipidome and proteome profiles were clearly distinguishable from post-placement ones. Metabolites in the pre-placement samples suggested an extinction of the energetic metabolism and a switch towards another source of fuelling (e.g., structural proteins). We were able to identify certain biomolecules with an excellent potential for PMI estimation, predominantly the biomolecules from the metabolomics block. Our findings suggest that, by targeting a combination of compounds with different postmortem stability, in the future we could be able to estimate both short PMIs, by using metabolites and lipids, and longer PMIs, by using proteins.

The symbiotic partnership between leaf-cutting ants and fungal cultivars processes plant biomass via ant fecal fluid mixed with chewed plant substrate before fungal degradation. Here we present a full proteome of the fecal fluid of Acromyrmex leaf-cutting ants, showing that most proteins function as biomass degrading enzymes and that ca. 85% are produced by the fungus and ingested, but not digested, by the ants. Hydrogen peroxide producing oxidoreductases were remarkably common in the proteome, inspiring us to test a scenario in which hydrogen peroxide reacts with iron to form reactive oxygen radicals after which oxidized iron is reduced by other fecal-fluid enzymes. Our biochemical assays confirmed that these so-called Fenton reactions do indeed take place in special substrate pellets, presumably to degrade plant cell wall polymers. This implies that the symbiotic partnership manages a combination of oxidative and enzymatic biomass degradation, an achievement that surpasses current human bioconversion technology.

DUX4 is a transcription factor whose misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). DUX4's transcriptional activity has been extensively characterized, but the DUX4-induced proteome remains undescribed. Here, we report concurrent measurement of RNA and protein levels in DUX4-expressing cells via RNA-seq and quantitative mass spectrometry. DUX4 transcriptional targets were robustly translated, confirming the likely clinical relevance of proposed FSHD biomarkers. However, a multitude of mRNAs and proteins exhibited discordant expression changes upon DUX4 expression. Our dataset revealed unexpected proteomic, but not transcriptomic, dysregulation of diverse molecular pathways, including Golgi apparatus fragmentation, as well as extensive post-transcriptional buffering of stress-response genes. Key components of RNA degradation machineries, including UPF1, UPF3B, and XRN1, exhibited suppressed protein, but not mRNA, levels, explaining the build-up of aberrant RNAs that characterizes DUX4-expressing cells. Our results provide a resource for the FSHD community and illustrate the importance of post-transcriptional processes in DUX4-induced pathology.

Master athletes (MAs) prove that preserving a high level of physical function up to very late in life is possible, but the mechanisms responsible for their high function remain unclear.