A proximity proteomics pipeline with improved reproducibility and throughput.

Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.

Cytoplasmic Shotgun Proteomic Points to Key Proteins and Pathways in Temozolomide-Resistant Glioblastoma Multiforme.

Temozolomide (TMZ) is the first line of chemotherapy to treat primary brain tumors of the type glioblastoma multiforme (GBM). TMZ resistance (TMZR) is one of the main barriers to successful treatment and is a principal factor in relapse, resulting in a poor median survival of 15 months. The present paper focuses on proteomic analyses of cytosolic fractions from TMZ-resistant (TMZR) LN-18 cells. The experimental workflow includes an easy, cost-effective, and reproducible method to isolate subcellular fraction of cytosolic (CYTO) proteins, mitochondria, and plasma membrane proteins for proteomic studies. For this study, enriched cytoplasmic fractions were analyzed in replicates by nanoflow liquid chromatography tandem high-resolution mass spectrometry (nLC-MS/MS), and proteins identified were quantified using a label-free approach (LFQ). Statistical analysis of control (CTRL) and temozolomide-resistant (TMZR) proteomes revealed proteins that appear to be differentially controlled in the cytoplasm. The functions of these proteins are discussed as well as their roles in other cancers and TMZ resistance in GBM. Key proteins are also described through biological processes related to gene ontology (GO), molecular functions, and cellular components. For protein-protein interactions (PPI), network and pathway involvement analyses have been performed, highlighting the roles of key proteins in the TMZ resistance phenotypes. This study provides a detailed insight into methods of subcellular fractionation for proteomic analysis of TMZ-resistant GBM cells and the potential to apply this approach to future large-scale studies. Several key proteins, protein-protein interactions (PPI), and pathways have been identified, underlying the TMZ resistance phenotype and highlighting the proteins' biological functions.

Subcellular proteomics insights into Alzheimer's disease development.

Alzheimer's disease (AD), one of the most common dementias, is a neurodegenerative disease characterized by cognitive impairment and decreased judgment function. The expected number of AD patient is increasing in the context of the world's advancing medical care and increasing human life expectancy. Since current molecular mechanism studies on AD pathogenesis are incomplete, there is no specific and effective therapeutic agent. Mass spectrometry (MS)-based unbiased proteomics studies provide an effective and comprehensive approach. Many advances have been made in the study of the mechanism, diagnostic markers, and drug targets of AD using proteomics. This paper focus on subcellular level studies, reviews studies using proteomics to study AD-associated mitochondrial dysfunction, synaptic, and myelin damage, the protein composition of amyloid plaques (APs) and neurofibrillary tangles (NFTs), changes in tissue extracellular vehicles (EVs) and exosome proteome, and the protein changes in ribosomes and lysosomes. The methods of sample separation and preparation and proteomic analysis as well as the main findings of these studies are involved. The results of these proteomics studies provide insights into the pathogenesis of AD and provide theoretical resource and direction for future research in AD, helping to identify new biomarkers and drugs targets for AD.

Quantitative proteomics of sperm tail in asthenozoospermic patients: exploring the molecular pathways affecting sperm motility.

Asthenozoospermia, characterized by low sperm motility, is one of the most common causes of male infertility. While many intrinsic and extrinsic factors are involved in the etiology of asthenozoospermia, the molecular basis of this condition remains unclear. Since sperm motility results from a complex flagellar structure, an in-depth proteomic analysis of the sperm tail can uncover mechanisms underlying asthenozoospermia. This study quantified the proteomic profile of 40 asthenozoospermic sperm tails and 40 controls using TMT-LC-MS/MS. Overall, 2140 proteins were identified and quantified where 156 proteins have not been described earlier in sperm tail. There were 409 differentially expressed proteins (250 upregulated and 159 downregulated) in asthenozoospermia which by far is the highest number reported earlier. Further, bioinformatics analysis revealed several biological processes, including mitochondrial-related energy production, oxidative phosphorylation (OXPHOS), citric acid cycle (CAC), cytoskeleton, stress response, and protein metabolism altered in asthenozoospermic sperm tail samples. Collectively, our findings reveal the importance of mitochondrial energy production and induced stress response as potential mechanisms involved in the loss of sperm motility in asthenozoospermia.

Recent Advances in the Prediction of Subcellular Localization of Proteins and Related Topics.

Prediction of subcellular localization of proteins from their amino acid sequences has a long history in bioinformatics and is still actively developing, incorporating the latest advances in machine learning and proteomics. Notably, deep learning-based methods for natural language processing have made great contributions. Here, we review recent advances in the field as well as its related fields, such as subcellular proteomics and the prediction/recognition of subcellular localization from image data.

Phosphoproteomics of primary AML patient samples reveals rationale for AKT combination therapy and p53 context to overcome selinexor resistance.

Acute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations.

Spatial Proteomics Reveals Differences in the Cellular Architecture of Antibody-Producing CHO and Plasma Cell-Derived Cells.

Most of the recombinant biotherapeutics employed today to combat severe illnesses, for example, various types of cancer or autoimmune diseases, are produced by Chinese hamster ovary (CHO) cells. To meet the growing demand of these pharmaceuticals, CHO cells are under constant development in order to enhance their stability and productivity. The last decades saw a shift from empirical cell line optimization toward rational cell engineering using a growing number of large omics datasets to alter cell physiology on various levels. Especially proteomics workflows reached new levels in proteome coverage and data quality because of advances in high-resolution mass spectrometry instrumentation. One type of workflow concentrates on spatial proteomics by usage of subcellular fractionation of organelles with subsequent shotgun mass spectrometry proteomics and machine learning algorithms to determine the subcellular localization of large portions of the cellular proteome at a certain time point. Here, we present the first subcellular spatial proteome of a CHO-K1 cell line producing high titers of recombinant antibody in comparison to the spatial proteome of an antibody-producing plasma cell-derived myeloma cell line. Both cell lines show colocalization of immunoglobulin G chains with chaperones and proteins associated in protein glycosylation within the endoplasmic reticulum compartment. However, we report differences in the localization of proteins associated to vesicle-mediated transport, transcription, and translation, which may affect antibody production in both cell lines. Furthermore, pairing subcellular localization data with protein expression data revealed elevated protein masses for organelles in the secretory pathway in plasma cell-derived MPC-11 (Merwin plasma cell tumor-11) cells. Our study highlights the potential of subcellular spatial proteomics combined with protein expression as potent workflow to identify characteristics of highly efficient recombinant protein-expressing cell lines. Data are available via ProteomeXchange with identifier PXD029115.

A Method to Estimate the Distribution of Proteins across Multiple Compartments Using Data from Quantitative Proteomics Subcellular Fractionation Experiments.

Knowledge of cellular location is key to understanding the biological function of proteins. One commonly used large-scale method to assign cellular locations is subcellular fractionation, followed by quantitative mass spectrometry to identify proteins and estimate their relative distribution among centrifugation fractions. In most of such subcellular proteomics studies, each protein is assigned to a single cellular location by comparing its distribution to those of a set of single-compartment reference proteins. However, in many cases, proteins reside in multiple compartments. To accurately determine the localization of such proteins, we previously introduced constrained proportional assignment (CPA), a method that assigns each protein a fractional residence over all reference compartments (Jadot Mol. Cell Proteomics 2017, 16(2), 194-212. 10.1074/mcp.M116.064527). In this Article, we describe the principles underlying CPA, as well as data transformations to improve accuracy of assignment of proteins and protein isoforms, and a suite of R-based programs to implement CPA and related procedures for analysis of subcellular proteomics data. We include a demonstration data set that used isobaric-labeling mass spectrometry to analyze rat liver fractions. In addition, we describe how these programs can be readily modified by users to accommodate a wide variety of experimental designs and methods for protein quantitation.

Determining protein polarization proteome-wide using physical dissection of individual Stentor coeruleus cells.

Cellular components are non-randomly arranged with respect to the shape and polarity of the whole cell.1-4 Patterning within cells can extend down to the level of individual proteins and mRNA.5,6 But how much of the proteome is actually localized with respect to cell polarity axes? Proteomics combined with cellular fractionation7-11 has shown that most proteins localize to one or more organelles but does not tell us how many proteins have a polarized localization with respect to the large-scale polarity axes of the intact cell. Genome-wide localization studies in yeast12-15 found that only a few percent of proteins have a localized position relative to the cell polarity axis defined by sites of polarized cell growth. Here, we describe an approach for analyzing protein distribution within a cell with a visibly obvious global patterning-the giant ciliate Stentor coeruleus.16,17 Ciliates, including Stentor, have highly polarized cell shapes with visible surface patterning.1,18 A Stentor cell is roughly 2 mm long, allowing a "proteomic dissection" in which microsurgery is used to separate cellular fragments along the anterior-posterior axis, followed by comparative proteomic analysis. In our analysis, 25% of the proteome, including signaling proteins, centrin/SFI proteins, and GAS2 orthologs, shows a polarized location along the cell's anterior-posterior axis. We conclude that a large proportion of all proteins are polarized with respect to global cell polarity axes and that proteomic dissection provides a simple and effective approach for spatial proteomics.

Nucleus and chloroplast: A necessary understanding to overcome heat stress in Pinus radiata.

The recovery and maintenance of plant homeostasis under stressful environments are complex processes involving organelle crosstalk for a coordinated cellular response. Here, we revealed through nuclear and chloroplast subcellular proteomics, biochemical cell profiles and targeted transcriptomics how chloroplasts and nuclei developed their responses under increased temperatures in a long-lived species (Pinus radiata). Parallel to photosynthetic impairment and reactive oxygen species production in the chloroplast, a DNA damage response was triggered in the nucleus followed by an altered chromatin conformation. In addition, in the nuclei, we found several proteins, such as HEMERA or WHIRLY, which change their locations from the chloroplasts to the nuclei carrying the stress message. Additionally, our data showed a deep rearrangement of RNA metabolism in both organelles, revealing microRNAs and AGO1 as potential regulators of the acclimation mechanisms. Altogether, our study highlights the synchronisation among the different stages required for thermotolerance acquisition in P. radiata, pointing out the role of chromatin conformation and posttranscriptional gene regulation in overcoming heat stress and assuring plant survival for the following years.

Methodologies for Measuring Protein Trafficking across Cellular Membranes.

Nearly all proteins are synthesized in the cytosol. The majority of this proteome must be trafficked elsewhere, such as to membranes, to subcellular compartments, or outside of the cell. Proper trafficking of nascent protein is necessary for protein folding, maturation, quality control and cellular and organismal health. To better understand cellular biology, molecular and chemical technologies to properly characterize protein trafficking (and mistrafficking) have been developed and applied. Herein, we take a biochemical perspective to review technologies that enable spatial and temporal measurement of protein distribution, focusing on both the most widely adopted methodologies and exciting emerging approaches.

Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum.

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes' protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.

Nitrogen Uptake in Plants: The Plasma Membrane Root Transport Systems from a Physiological and Proteomic Perspective.

Nitrogen nutrition in plants is a key determinant in crop productivity. The availability of nitrogen nutrients in the soil, both inorganic (nitrate and ammonium) and organic (urea and free amino acids), highly differs and influences plant physiology, growth, metabolism, and root morphology. Deciphering this multifaceted scenario is mandatory to improve the agricultural sustainability. In root cells, specific proteins located at the plasma membrane play key roles in the transport and sensing of nitrogen forms. This review outlines the current knowledge regarding the biochemical and physiological aspects behind the uptake of the individual nitrogen forms, their reciprocal interactions, the influences on root system architecture, and the relations with other proteins sustaining fundamental plasma membrane functionalities, such as aquaporins and H+-ATPase. This topic is explored starting from the information achieved in the model plant Arabidopsis and moving to crops in agricultural soils. Moreover, the main contributions provided by proteomics are described in order to highlight the goals and pitfalls of this approach and to get new hints for future studies.

Ubiquitin carboxyl-terminal hydrolase isozyme L1/UCHL1 suppresses epithelial-mesenchymal transition and is under-expressed in cadmium-transformed human bronchial epithelial cells.

Cadmium (Cd), a highly toxic heavy metal, is widespreadly distributed in the environment. Chronic exposure to Cd is associated with the development of several diseases including cancers. Over the decade, many researches have been carried on various models to examine the acute effects of Cd; yet, limited knowledge is known about the long-term Cd exposure, especially in the human lung cells. Previously, we showed that chronic Cd-exposed human bronchial epithelial BEAS-2B cells exhibited transformed cell properties, such as anchorage-independent growth, augmented cell migration, and epithelial-mesenchymal transition (EMT). To study these Cd-transformed cells more comprehensively, here, we further characterized their subproteomes. Overall, a total of 63 differentially expressed proteins between Cd-transformed and passage-matched control cells among the five subcellular fractions (cytoplasmic, membrane, nuclear-soluble, chromatin-bound, and cytoskeletal) were identified by mass spectrometric analysis and database searching. Interestingly, we found that the thiol protease ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) is one of the severely downregulated proteins in the Cd-transformed cells. Notably, the EMT phenotype of Cd-transformed cells can be suppressed by forced ectopic expression of UCHL1, suggesting UCHL1 as a crucial modulator in the maintenance of the proper differentiation status in lung epithelial cells. Since EMT is considered as a critical step during malignant cell transformation, finding novel cellular targets that can antagonize this transition may lead to more efficient strategies to inhibit cancer development. Our data report for the first time that UCHL1 may play a function in the suppression of EMT in Cd-transformed human lung epithelial cells, indicating that UCHL1 might be a new therapeutic target for chronic Cd-induced carcinogenesis. Graphical abstract.

Proteomic comparison of different synaptosome preparation procedures.

Synaptosomes are frequently used research objects in neurobiology studies focusing on synaptic transmission as they mimic several aspects of the physiological synaptic functions. They contain the whole apparatus for neurotransmission, the presynaptic nerve ending with synaptic vesicles, synaptic mitochondria and often a segment of the postsynaptic membrane along with the postsynaptic density is attached to its outer surface. As being artificial functional organelles, synaptosomes are viable for several hours, retain their activity, membrane potential, and capable to store, release, and reuptake neurotransmitters. Synaptosomes are ideal subjects for proteomic analysis. The recently available separation and protein detection techniques can cope with the reduced complexity of the organelle and enable the simultaneous qualitative and quantitative analysis of thousands of proteins shaping the structural and functional characteristics of the synapse. Synaptosomes are formed during the homogenization of nervous tissue in the isoosmotic milieu and can be isolated from the homogenate by various approaches. Each enrichment method has its own benefits and drawbacks and there is not a single method that is optimal for all research purposes. For a proper proteomic experiment, it is desirable to preserve the native synaptic structure during the isolation procedure and keep the degree of contamination from other organelles or cell types as low as possible. In this article, we examined five synaptosome isolation methods from a proteomic point of view by the means of electron microscopy, Western blot, and liquid chromatography-mass spectrometry to compare their efficiency in the isolation of synaptosomes and depletion of contaminating subcellular structures. In our study, the different isolation procedures led to a largely overlapping pool of proteins with a fairly similar distribution of presynaptic, active zone, synaptic vesicle, and postsynaptic proteins; however, discrete differences were noticeable in individual postsynaptic proteins and in the number of identified transmembrane proteins. Much pronounced variance was observed in the degree of contamination with mitochondrial and glial structures. Therefore, we suggest that in selecting the appropriate isolation method for any neuroproteomics experiment carried out on synaptosomes, the degree and sort/source of contamination should be considered as a primary aspect.

Subcellular Proteomics in Conifers: Purification of Nuclei and Chloroplast Proteomes.

The complexity of the plant cell proteome, exhibiting thousands of proteins whose abundance varies in several orders of magnitude, makes impossible to cover most of the plant proteins using standard shotgun-based approaches. Despite this general description of plant proteomes, the complexity is not a big issue (current protocols and instrumentation allow for the identification of several thousand proteins per injection), low or medium abundant proteins cannot be detected most of times, being necessary to fraction or perform targeted analyses in order to detect and quantify them. Among fractioning choices, cell fractioning in its different organelles is a good strategy for gaining not only a deeper coverage of the proteome but also the basis for understanding organelle function, protein dynamics, and trafficking within the cell, as nuclear and chloroplast communication. This approach is used routinely in many labs working with model species; however, the available protocols focusing on tree species are scarce. In this chapter, we provide a simple but robust protocol for isolating nuclei and chloroplasts in pine needles that is fully compatible with later mass spectrometry-based proteome analysis.

Isolation, Purity Assessment, and Proteomic Analysis of Endoplasmic Reticulum.

Subcellular proteomics include, in its experimental workflow, steps aimed at purifying organelles. The purity of the subcellular fraction should be assessed before mass spectrometry analysis, in order to confidently conclude the presence of associated specific proteoforms, deepening the knowledge of its biological function. In this chapter, a protocol for isolating endoplasmic reticulum (ER) and purity assessment is reported, and it precedes the proteomic analysis through a gel-free/label-free proteomic approach. Dysfunction of quality-control mechanisms of protein metabolism in ER leads to ER stress. Additionally, ER, which is a calcium-storage organelle, is responsible for signaling and homeostatic function, and calcium homeostasis is required for plant tolerance. With such predominant cell functions, effective protocols to fractionate highly purified ER are needed. Here, isolation methods and purity assessments of ER are described. In addition, a gel-free/label-free proteomic approach of ER is presented.

Subcellular proteome profiles of different latex fractions revealed washed solutions from rubber particles contain crucial enzymes for natural rubber biosynthesis.

Rubber particle (RP) is a specific organelle for natural rubber biosynthesis (NRB) and storage in rubber tree Hevea brasiliensis. NRB is processed by RP membrane-localized proteins, which were traditionally purified by repeated washing. However, we noticed many proteins in the discarded washing solutions (WS) from RP. Here, we compared the proteome profiles of WS, C-serum (CS) and RP by 2-DE, and identified 233 abundant proteins from WS by mass spectrometry. Many spots on 2-DE gels were identified as different protein species. We further performed shotgun analysis of CS, WS and RP and identified 1837, 1799 and 1020 unique proteins, respectively. Together with 2-DE, we finally identified 1825 proteins from WS, 246 were WS-specific. These WS-specific proteins were annotated in Gene Ontology, indicating most abundant pathways are organic substance metabolic process, protein degradation, primary metabolic process, and energy metabolism. Protein-protein interaction analysis revealed these WS-specific proteins are mainly involved in ribosomal metabolism, proteasome system, vacuolar protein sorting and endocytosis. Label free and Western blotting revealed many WS-specific proteins and protein complexes are crucial for NRB initiation. These findings not only deepen our understanding of WS proteome, but also provide new evidences on the roles of RP membrane proteins in NRB. SIGNIFICANCE: Natural rubber is stored in rubber particle from the rubber tree. Rubber particles were traditionally purified by repeated washing, but many proteins were identified from the washing solutions (WS). We obtained the first visualization proteome profiles with 1825 proteins from WS, including 246 WS-specific ones. These WS proteins contain almost all enzymes for polyisoprene initiation and may play important roles in rubber biosynthesis.

Subcellular Proteomics: Application to Elucidation of Flooding-Response Mechanisms in Soybean.

Soybean, which is rich in protein and oil, is cultivated in several climatic zones; however, its growth is markedly decreased by flooding. Proteomics is a useful tool for understanding the flooding-response mechanism in soybean. Subcellular proteomics has the potential to elucidate localized cellular responses and investigate communications among subcellular components during plant growth and during stress. Under flooding, proteins related to signaling, stress and the antioxidative system are increased in the plasma membrane; scavenging enzymes for reactive-oxygen species are suppressed in the cell wall; protein translation is suppressed through inhibition of proteins related to preribosome biogenesis and mRNA processing in the nucleus; levels of proteins involved in the electron transport chain are reduced in the mitochondrion; and levels of proteins related to protein folding are decreased in the endoplasmic reticulum. This review discusses the advantages of a gel-free/label-free proteomic technique and methods of plant subcellular purification. It also summarizes cellular events in soybean under flooding and discusses future prospects for generation of flooding-tolerant soybean.

Network based subcellular proteomics in monocyte membrane revealed novel candidate genes involved in osteoporosis.

In this study, label-free-based quantitative subcellular proteomics integrated with network analysis highlighted several candidate genes including P4HB, ITGB1, CD36, and ACTN1 that may be involved in osteoporosis. All of them are predicted as significant membrane proteins with high confidence and enriched in bone-related biological process. The results were further verified in transcriptomic and genomic levels. INTRODUCTION: Osteoporosis is a metabolic bone disease mainly characterized by low bone mineral density (BMD). As the precursors of osteoclasts, peripheral blood monocytes (PBMs) are supported to be important candidates for identifying genes related to osteoporosis. We performed subcellular proteomics study to identify significant membrane proteins that involved in osteoporosis. METHODS: To investigate the association between monocytes, membrane proteins, and osteoporosis, we performed label-free quantitative subcellular proteomics in 59 male subjects with discordant BMD levels, with 30 high vs. 29 low BMD subjects. Subsequently, we performed integrated gene enrichment analysis, functional annotation, and pathway and network analysis based on multiple bioinformatics tools. RESULTS: A total of 1070 membrane proteins were identified and quantified. By comparing the proteins' expression level, we found 36 proteins that were differentially expressed between high and low BMD groups. Protein localization prediction supported the notion that the differentially expressed proteins, P4HB (p = 0.0021), CD36 (p = 0.0104), ACTN1 (p = 0.0381), and ITGB1 (p = 0.0385), are significant membrane proteins. Functional annotation and pathway and network analysis highlighted that P4HB, ITGB1, CD36, and ACTN1 are enriched in osteoporosis-related pathways and terms including "ECM-receptor interaction," "calcium ion binding," "leukocyte transendothelial migration," and "reduction of cytosolic calcium levels." Results from transcriptomic and genomic levels provided additional supporting evidences. CONCLUSION: Our study strongly supports the significance of the genes P4HB, ITGB1, CD36, and ACTN1 to the etiology of osteoporosis risk.