Selective Roles of Vertebrate PCF11 in Premature and Full-Length Transcript Termination.

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3' mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.

Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints.

N-linked glycosylation is a biologically important protein modification, but only a small fraction of modification sites have been mapped. We developed a "filter aided sample preparation" (FASP)-based method in which glycopeptides are enriched by binding to lectins on the top of a filter and mapped 6367 N-glycosylation sites on 2352 proteins in four mouse tissues and blood plasma using high-accuracy mass spectrometry. We found 74% of known mouse N-glycosites and discovered an additional 5753 sites on a diverse range of proteins. Sites almost always have the N-!P-[S|T]-!P (where !P is not proline) and rarely the N-X-C motif or nonconsensus sequences. Combining the FASP approach with analysis of subcellular glycosite localization reveals that the sites always orient toward the extracellular space or toward the lumen of ER, Golgi, lysosome, or peroxisome. The N-glycoproteome contains a plethora of modification sites on factors important in development, organ-specific functions, and disease.

Interlaboratory Variability in the Madin-Darby Canine Kidney Cell Proteome.

Madin-Darby canine kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate transepithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits, and as such, laboratory-to-laboratory variability in drug permeability assessments is observed. Consequently, in vitro-in vivo extrapolation (IVIVE) approaches using permeability and/or transporter activity data require calibration. A comprehensive proteomic quantification of 11 filter-grown parental or mock-transfected MDCK monolayers from 8 different pharmaceutical laboratories using the total protein approach (TPA) is provided. The TPA enables estimations of key morphometric parameters such as monolayer cellularity and volume. Overall, metabolic liability to xenobiotics is likely to be limited for MDCK cells due to the low expression of required enzymes. SLC16A1 (MCT1) was the highest abundant SLC transporter linked to xenobiotic activity, while ABCC4 (MRP4) was the highest abundant ABC transporter. Our data supports existing findings that claudin-2 levels may be linked to tight junction modulation, thus impacting trans-epithelial resistance. This unique database provides data on more than 8000 protein copy numbers and concentrations, thus allowing an in-depth appraisal of the control monolayers used in each laboratory.

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis.

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.

Analysis of the Site-Specific Myoglobin Modifications in the Melibiose-Derived Novel Advanced Glycation End-Product.

MAGE (melibiose-derived advanced glycation end-product) is the glycation product generated in the reaction of a model protein with melibiose. The in vivo analog accumulates in several tissues; however, its origin still needs explanation. In vitro MAGE is efficiently generated under dry conditions in contrast to the reaction carried in an aqueous solvent. Using liquid chromatography coupled with mass spectrometry, we analyzed the physicochemical properties and structures of myoglobin glycated with melibiose under different conditions. The targeted peptide analysis identified structurally different AGEs, including crosslinking and non-crosslinking modifications associated with lysine, arginine, and histidine residues. Glycation in a dry state was more efficient in the formation of structures containing an intact melibiose moiety (21.9%) compared to glycation under aqueous conditions (15.6%). The difference was reflected in characteristic fluorescence that results from protein structural changes and impact on a heme group of the model myoglobin protein. Finally, our results suggest that the formation of in vitro MAGE adduct is initiated by coupling melibiose to a model myoglobin protein. It is confirmed by the identification of intact melibiose moieties. The intermediate glycation product can further rearrange towards more advanced structures, including cross-links. This process can contribute to a pool of AGEs accumulating locally in vivo and affecting tissue biology.

Absolute quantitative proteomics using the total protein approach to identify novel clinical immunohistochemical markers in renal neoplasms.

BACKGROUND: Renal neoplasms encompass a variety of malignant and benign tumors, including many with shared characteristics. The diagnosis of these renal neoplasms remains challenging with currently available tools. In this work, we demonstrate the total protein approach (TPA) based on high-resolution mass spectrometry (MS) as a tool to improve the accuracy of renal neoplasm diagnosis. METHODS: Frozen tissue biopsies of human renal tissues [clear cell renal cell carcinoma (n = 7), papillary renal cell carcinoma (n = 5), chromophobe renal cell carcinoma (n = 5), and renal oncocytoma (n = 5)] were collected for proteome analysis. Normal adjacent renal tissue (NAT, n = 5) was used as a control. Proteins were extracted and digested using trypsin, and the digested proteomes were analyzed by label-free high-resolution MS (nanoLC-ESI-HR-MS/MS). Quantitative analysis was performed by comparison between protein abundances of tumors and NAT specimens, and the label-free and standard-free TPA was used to obtain absolute protein concentrations. RESULTS: A total of 205 differentially expressed proteins with the potential to distinguish the renal neoplasms were found. Of these proteins, a TPA-based panel of 24, including known and new biomarkers, was selected as the best candidates to differentiate the neoplasms. As proof of concept, the diagnostic potential of PLIN2, TUBB3, LAMP1, and HK1 was validated using semi-quantitative immunohistochemistry with a total of 128 samples assessed on tissue micro-arrays. CONCLUSIONS: We demonstrate the utility of combining high-resolution MS and the TPA as potential new diagnostic tool in the pathology of renal neoplasms. A similar TPA approach may be implemented in any cancer study with solid biopsies.

Compatibility of Distinct Label-Free Proteomic Workflows in Absolute Quantification of Proteins Linked to the Oocyte Quality in Human Follicular Fluid.

We present two separate label-free quantitative workflows based on different high-resolution mass spectrometers and LC setups, which are termed after the utilized instrument: Quad-Orbitrap (nano-LC) and Triple Quad-TOF (micro-LC) and their directed adaptation toward the analysis of human follicular fluid proteome. We identified about 1000 proteins in each distinct workflow using various sample preparation methods. With assistance of the Total Protein Approach, we were able to obtain absolute protein concentrations for each workflow. In a pilot study of twenty samples linked to diverse oocyte quality status from four donors, 455 and 215 proteins were quantified by the Quad-Orbitrap and Triple Quad-TOF workflows, respectively. The concentration values obtained from both workflows correlated to a significant degree. We found reasonable agreement of both workflows in protein fold changes between tested groups, resulting in unified lists of 20 and 22 proteins linked to oocyte maturity and blastocyst development, respectively. The Quad-Orbitrap workflow was best suited for an in-depth analysis without the need of extensive fractionation, especially of low abundant proteome, whereas the Triple Quad-TOF workflow allowed a more robust approach with a greater potential to increase in effectiveness with the growing number of analyzed samples after the initial effort of building a comprehensive spectral library.

Absolute Proteome Analysis of Hippocampus, Cortex and Cerebellum in Aged and Young Mice Reveals Changes in Energy Metabolism.

Aging is associated with a general decline of cognitive functions, and it is widely accepted that this decline results from changes in the expression of proteins involved in regulation of synaptic plasticity. However, several lines of evidence have accumulated that suggest that the impaired function of the aged brain may be related to significant alterations in the energy metabolism. In the current study, we employed the label-free "Total protein approach" (TPA) method to focus on the similarities and differences in energy metabolism proteomes of young (1-month-old) and aged (22-month-old) murine brains. We quantified over 7000 proteins in each of the following three analyzed brain structures: the hippocampus, the cerebral cortex and the cerebellum. To the best of our knowledge, this is the most extensive quantitative proteomic description of energy metabolism pathways during the physiological aging of mice. The analysis demonstrates that aging does not significantly affect the abundance of total proteins in the studied brain structures, however, the levels of proteins constituting energy metabolism pathways differ significantly between young and aged mice.

Ultrasonic-Based Filter Aided Sample Preparation as the General Method to Sample Preparation in Proteomics.

We propose a new high-throughput ultrafast method for large-scale proteomics approaches by speeding up the classic filter aided sample preparation protocol, FASP, from overnight to 2.5 h. Thirty-six samples can be treated in 2.5 h, and the method is scalable to 96-well plate-based pipelines. After a modification of the FASP-tube, the steps of protein reduction, protein alkylation, and protein digestion of complex proteomes are done in just 5.25 min, each one under the effects of an ultrasonic field (7 cycles: 30 s on and 15 s off). The new method was compared to the standard overnight digestion FASP protocol, and no statistical differences were found for more than 92.4%, 92%, and 93.3% of the proteins identified by studying the proteome of E. coli, mouse brain, and mouse liver tissue samples, respectively. Furthermore, the successful relative label-free quantification of four spiked proteins in E. coli samples, BSA, ß-lactoglobulin, α-casein, and α-lactalbumin, was achieved, using either the ultrasonic-based FASP protocol or the classic overnight one. The new US-FASP method matches the analytical minimalism rules as time, cost, sample requirement, reagent consumption, energy requirements, and production of waste products are reduced to a minimum while maintaining high sample throughput in a robust manner as all of the advantages of the filter aided sample preparation protocol are maintained.

Cell-type-resolved proteomic analysis of the human liver.

BACKGROUND & AIMS: The human liver functions through a complex interplay between parenchymal and non-parenchymal cells. Mass spectrometry-based proteomic analysis of intact tissue has provided an in-depth view of the human liver proteome. However, the predominance of parenchymal cells (hepatocytes) means that the total tissue proteome mainly reflects hepatocyte expression. Here we therefore set out to analyse the proteomes of the major parenchymal and non-parenchymal cell types in the human liver. METHODS: We applied quantitative label-free proteomic analysis on the major cell types of the human liver: hepatocytes, liver endothelial cells, Kupffer cells and hepatic stellate cells. RESULTS: We identified 9791 proteins, revealing distinct protein expression profiles across cell types, whose in vivo relevance was shown by the presence of cell-type-specific proteins. Analysis of proteins related to the immune system indicated that mechanisms of immune-mediated liver injury include the involvement of several cell types. Furthermore, in-depth investigation of proteins related to the absorption, distribution, metabolism, excretion and toxicity (ADMET) of xenobiotics showed that ADMET-related tasks are not exclusively confined to hepatocytes, and that non-parenchymal cells may contribute to drug transport and metabolism. CONCLUSIONS: Overall, the data we provide constitute a unique resource for exploring the proteomes of the major types of human liver cells, which will facilitate an improved understanding of the human liver in health and disease.

Mapping N-glycosylation sites across seven evolutionarily distant species reveals a divergent substrate proteome despite a common core machinery.

N-linked glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteomes in nonmammalian systems. Here, we measure N-glycoproteomes of the major model organisms Arabidopsis thaliana, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio, representatively spanning the eukaryotic domain of life. The number of detected N-glycosylation sites varied between 425 in fission yeast, 516 in budding yeast, 1,794 in worm, 2,186 in plant, 2,229 in fly, and 2,254 in zebrafish. We find that all eukaryotic N-glycoproteomes have invariant characteristics including sequence recognition patterns, structural constraints, and subcellular localization. However, a surprisingly large percentage of the N-glycoproteome evolved after the phylogenetic divergences between plants, fungi, nematodes, insects, and vertebrates. Many N-glycosylated proteins coevolved with the rise of extracellular processes that are specific within corresponding phylogenetic groups and essential for organismal development, body growth, and organ formation.

A dual role of linker histone H1.4 Lys 34 acetylation in transcriptional activation.

The linker histone H1 is a key player in chromatin organization, yet our understanding of the regulation of H1 functions by post-translational modifications is very limited. We provide here the first functional characterization of H1 acetylation. We show that H1.4K34 acetylation (H1.4K34ac) is mediated by GCN5 and is preferentially enriched at promoters of active genes, where it stimulates transcription by increasing H1 mobility and recruiting a general transcription factor. H1.4K34ac is dynamic during spermatogenesis and marks undifferentiated cells such as induced pluripotent stem (iPS) cells and testicular germ cell tumors. We propose a model for H1.4K34ac as a novel regulator of chromatin function with a dual role in transcriptional activation.

Multiple-Enzyme-Digestion Strategy Improves Accuracy and Sensitivity of Label- and Standard-Free Absolute Quantification to a Level That Is Achievable by Analysis with Stable Isotope-Labeled Standard Spiking.

Quantification of individual proteins is an essential task in understanding biological processes. For example, determination of concentrations of proteins transporting and metabolizing xenobiotics is a prerequisite for drug disposition predictions in humans based on in vitro data. So far, this task has frequently been accomplished by targeted proteomics. This type of analyses requires preparation of stable isotope labeled standards for each protein of interest. The selection of appropriate standard peptides is usually tedious and the number of proteins that can be studied in a single experiment by these approaches is limited. In addition, incomplete digestion of proteins often affects the accuracy of the quantification. To circumvent these constrains in proteomic protein quantification, label- and standard-free approaches, such as "total protein approach" (TPA) have been proposed. Here we directly compare an approach using stable isotope labeled (SIL) standards and TPA for quantification of transporters and enzymes in human liver samples within the same LC-MS/MS runs. We show that TPA is a convenient alternative to SIL-based methods. Optimization of the sample preparation beyond commonly used single tryptic digestion, by adding consecutive cleavage steps, improves accuracy and reproducibility of the TPA method to a level, which is achievable by analysis using stable isotope-labeled standard spiking.

A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes.

Primary human hepatocytes are used in all facets of liver research, from in vitro studies of xenobiotic disposition and toxicity to the clinical management of liver failure. Unfortunately, cellular stress during isolation and cryopreservation causes a highly unpredictable loss of the ability to attach and form cell-matrix and cell-cell interactions. Reasoning that this problem could be mitigated at the post-thawing stage, we applied label-free quantitative global proteomics to analyze differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, ultimately leading to apoptosis activation. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately post-thawing, which suggested the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. Brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored attachment ability and promoted a differentiated morphology, as well as formation of 3D spheroids. Further, Z-VAD-FMK treatment restored metabolic and transport functions, with maintained sensitivity to hepatotoxic insults. Altogether, this study shows that differentiation and function of suboptimal human hepatocytes can be restored after cryopreservation, thus markedly increasing the availability of these precious cells.

In-Depth Quantitative Proteomic Analysis of Trophozoites and Pseudocysts of Trichomonas vaginalis.

Trichomonas vaginalis is a sexually transmitted anaerobic parasite that infects humans causing trichomoniasis, a common and ubiquitous sexually transmitted disease. The life cycle of this parasite possesses a trophozoite form without a cystic stage. However, the presence of nonproliferative and nonmotile, yet viable and reversible spherical forms with internalized flagella, denominated pseudocysts, has been commonly observed for this parasite. To understand the mechanisms involved in the formation of pseudocysts, we performed a mass spectrometry-based high-throughput quantitative proteomics study using a label-free approach and functional assays by biochemical and flow cytometric methods. We observed that the morphological transformation of trophozoite to pseudocysts is coupled to (i) a metabolic shift toward a less glycolytic phenotype; (ii) alterations in the abundance of hydrogenosomal iron-sulfur cluster (ISC) assembly machinery; (iii) increased abundance of regulatory particles of the ubiquitin-proteasome system; (iv) significant alterations in proteins involved in adhesion and cytoskeleton reorganization; and (v) arrest in G2/M phase associated with alterations in the abundance of regulatory proteins of the cell cycle. These data demonstrate that pseudocysts experience important physiological and structural alterations for survival under unfavorable environmental conditions.

Aging-associated changes in hippocampal glycogen metabolism in mice. Evidence for and against astrocyte-to-neuron lactate shuttle.

Lactate derived from astrocytic glycogen has been shown to support memory formation in hippocampi of young animals, inhibiting it in old animals. Here we show, using quantitative mass spectrometry-based proteomics, immunofluorescence, and qPCR that aging is associated with an increase of glycogen metabolism enzymes concentration and shift in their localization from astrocytes to neurons. These changes are accompanied with reorganization of hippocampal energy metabolism which is manifested by elevated capacity of aging neurons to oxidize glucose in glycolysis and mitochondria, and decreased ability for fatty acids utilization. Our observations suggest that astrocyte-to-neuron lactate shuttle may operate in young hippocampi, however, during aging neurons become independent on astrocytic lactate and the metabolic crosstalk between the brain's cells is disrupted.

Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids.

Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability ( Fic) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower Fic. The induction of NL did not further increase drug binding but led to altered Fic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.

Translating Proteomic Into Functional Data: An High Mobility Group A1 (HMGA1) Proteomic Signature Has Prognostic Value in Breast Cancer.

Cancer is a very heterogeneous disease, and biological variability adds a further level of complexity, thus limiting the ability to identify new genes involved in cancer development. Oncogenes whose expression levels control cell aggressiveness are very useful for developing cellular models that permit differential expression screenings in isogenic contexts. HMGA1 protein has this unique property because it is a master regulator in breast cancer cells that control the transition from a nontumorigenic epithelial-like phenotype toward a highly aggressive mesenchymal-like one. The proteins extracted from HMGA1-silenced and control MDA-MB-231 cells were analyzed using label-free shotgun mass spectrometry. The differentially expressed proteins were cross-referenced with DNA microarray data obtained using the same cellular model and the overlapping genes were filtered for factors linked to poor prognosis in breast cancer gene expression meta-data sets, resulting in an HMGA1 protein signature composed of 21 members (HRS, HMGA1 reduced signature). This signature had a prognostic value (overall survival, relapse-free survival, and distant metastasis-free survival) in breast cancer. qRT-PCR, Western blot, and immunohistochemistry analyses validated the link of three members of this signature (KIFC1, LRRC59, and TRIP13) with HMGA1 expression levels both in vitro and in vivo and wound healing assays demonstrated that these three proteins are involved in modulating tumor cell motility. Combining proteomic and genomic data with the aid of bioinformatic tools, our results highlight the potential involvement in neoplastic transformation of a restricted list of factors with an as-yet-unexplored role in cancer. These factors are druggable targets that could be exploited for the development of new, targeted therapeutic approaches in triple-negative breast cancer.

Quantitative Analysis of Human Red Blood Cell Proteome.

Red blood cells (RBCs) are the most abundant cell type in the human body. RBCs and, in particular, their plasma membrane composition have been extensively studied for many years. During the past decade proteomics studies have extended our knowledge on RBC composition; however, these studies did not provide quantitative insights. Here we report a large-scale proteomics investigation of RBCs and their "white ghost" membrane fraction. Samples were processed using the multienzyme digestion filter-aided sample preparation (MED-FASP) and analyzed using Q-Exactive mass spectrometer. Protein abundances were computed using the total protein approach (TPA). The validation of the data with stable isotope-labeled peptide-based protein quantification followed. Our in-depth analysis resulted in the identification of 2650 proteins, of which 1890 occurred at more than 100 copies per cell. We quantified 41 membrane transporter proteins spanning an abundance range of five orders of magnitude. Some of these, including the drug transporter ABCA7 and choline transporters SLC44A1 and SLC44A2, have not previously been identified in RBC membranes. Comparison of protein copy numbers assessed by proteomics showed a good correlation with literature data; however, abundances of several proteins were not consistent with the classical references. Because we validated our findings by a targeted analysis using labeled standards, our data suggest that some older reference data from a variety of biochemical approaches are inaccurate. Our study provides the first "in-depth" quantitative analysis of the RBC proteome and will promote future studies of erythrocyte structure, functions, and disease.

Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism.

BACKGROUND: It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein-fibrinogen and fibrin that forms a plasma clot. METHODS: The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC-MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results. RESULTS: Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC-MS/MS. CONCLUSIONS: The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.