Multi-Omic Analysis of Esophageal Adenocarcinoma Uncovers Candidate Therapeutic Targets and Cancer-Selective Posttranscriptional Regulation.

Efforts to address the poor prognosis associated with esophageal adenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome and which somatic variants are of importance for the disease phenotype. We performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance using tissue-matched RNA-seq and proteomic data from seven patients and further integrated these data with a cohort of EAC RNA-seq data (n = 264 patients), EAC whole-genome sequencing (n = 454 patients), and external published datasets. We quantified protein expression from 5879 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-selective expression were identified, including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated protein and RNA abundance, suggesting posttranscriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation, suggesting post-transcriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be overexpressed at the protein level in EAC compared to adjacent normal tissues with an EAC-selective, post-transcriptional mechanism of regulation of protein abundance proposed. Integrated analysis of proteome, transcriptome, and genome in EAC has revealed several genes with tumor-selective, posttranscriptional regulation of protein expression, which may be an exploitable vulnerability.

Exploiting ion-mobility mass spectrometry for unraveling proteome complexity.

Ion mobility spectrometry-mass spectrometry (IMS-MS) is experiencing rapid growth in proteomic studies, driven by its enhancements in dynamic range and throughput, increasing the quantitation precision, and the depth of proteome coverage. The core principle of ion mobility spectrometry is to separate ions in an inert gas under the influence of an electric field based on differences in drift time. This minireview provides an introduction to IMS operation modes and a description of advantages and limitations is presented. Moreover, the principles of trapped IMS-MS (TIMS-MS), including parallel accumulation-serial fragmentation are discussed. Finally, emerging applications linked to TIMS focusing on sample throughput (in clinical proteomics) and sensitivity (single-cell proteomics) are reviewed, and the possibilities of intact protein analysis are discussed.

YBEY is an essential biogenesis factor for mitochondrial ribosomes.

Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.

A novel proline substitution (Arg201Pro) in alpha helix 8 of TMEM98 causes autosomal dominant nanophthalmos-4, closed angle glaucoma and attenuated visual acuity.

Nanophthalmos-4 is a rare autosomal dominant disorder caused by two known variations in TMEM98. An Austrian Caucasian pedigree was identified suffering from nanophthalmos and late onset angle-closure glaucoma and premature loss of visual acuity. Whole exome sequencing identified segregation of a c.602G > C transversion in TMEM98 (p.Arg201Pro) as potentially causative. A protein homology model generated showed a TMEM98 structure comprising α4, α5/6, α7 and α8 antiparallel helix bundles and two predicted transmembrane domains in α1 and α7 that have been confirmed in vitro. Both p.Arg201Pro and the two missense variations representing proline insertions identified previously to cause nanophthalmos-4 (p.Ala193Pro and p.His196Pro) are located in the charge polarized helix α8 (p.183-p210). Stability of the C-terminal alpha helical structure of TMEM98 is therefore essential to prevent the development of human nanophthalmos-4. Precise molecular diagnosis could lead to the development of tailored therapies for patients with orphan ocular disease.

Platelet-Rich Fibrin Increases BMP2 Expression in Oral Fibroblasts via Activation of TGF-ß Signaling.

Solid platelet-rich fibrin (PRF), consisting of coagulated plasma from fractionated blood, has been proposed to be a suitable carrier for recombinant bone morphogenetic protein 2 (BMP2) to target mesenchymal cells during bone regeneration. However, whether solid PRF can increase the expression of BMPs in mesenchymal cells remains unknown. Proteomics analysis confirmed the presence of TGF-ß1 but not BMP2 in PRF lysates. According to the existing knowledge of recombinant TGF-ß1, we hypothesized that PRF can increase BMP2 expression in mesenchymal cells. To test this hypothesis, we blocked TGF-ß receptor 1 kinase with SB431542 in gingival fibroblasts exposed to PRF lysates. RT-PCR and immunoassays confirmed that solid PRF lysates caused a robust SB431542-dependent increase in BMP2 expression in gingival fibroblasts. Additionally, fractions of liquid PRF, namely platelet-poor plasma (PPP) and the buffy coat (BC) layer, but not heat-denatured PPP (Alb-gel), greatly induced the expression of BMP2 in gingival fibroblasts. Even though PRF has no detectable BMPs, PRF lysates similar to recombinant TGF-ß1 had the capacity to provoke canonical BMP signaling, as indicated by the nuclear translocation of Smad1/5 and the increase in its phosphorylation. Taken together, our data suggest that PRF can activate TGF-ß receptor 1 kinase and consequently induce the production of BMP2 in cells of the mesenchymal lineage.

Quantitative proteome analysis of Merkel cell carcinoma cell lines using SILAC.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumour of the skin with growing incidence. To better understand the biology of this malignant disease, immortalized cell lines are used in research for in vitro experiments. However, a comprehensive quantitative proteome analysis of these cell lines has not been performed so far. METHODS: Stable isotope labelling by amino acids in cell culture (SILAC) was applied to six MCC cell lines (BroLi, MKL-1, MKL-2, PeTa, WaGa, and MCC13). Following tryptic digest of labelled proteins, peptides were analysed by mass spectrometry. Proteome patterns of MCC cell lines were compared to the proteome profile of an immortalized keratinocyte cell line (HaCaT). RESULTS: In total, 142 proteins were upregulated and 43 proteins were downregulated. Altered proteins included mitoferrin-1, histone H2A type 1-H, protein-arginine deiminase type-6, heterogeneous nuclear ribonucleoproteins A2/B1, protein SLX4IP and clathrin light chain B. Furthermore, several proteins of the histone family and their variants were highly abundant in MCC cell lines. CONCLUSIONS: The results of this study present a new protein map of MCC and provide deeper insights in the biology of MCC. Data are available via ProteomeXchange with identifier PXD008181.

Human chorionic gonadotrophin pharmaceutical formulations of urinary origin display high levels of contaminant proteins-A label-free quantitation proteomics study.

To determine whether there is a measurable protein background in different formulations of urinary and recombinant human chorionic gonadotropin (hCG). Primary outcome measures: identification of contaminant proteins in urinary-derived formulations of hCG; secondary outcome measures: quantitative values of contaminant proteins in different batches of urinary -derived hCG formulations. It was found that urinary-derived batches have high presence of contaminant proteins beside the active substance. The relative amount of contaminant proteins and hCG differs strongly between different batches.

Serum proteomic pattern in female stress urinary incontinence.

The pathophysiology of Stress Urinary Incontinence (SUI) is poorly understood. The aim of this study was to identify the serum proteomic profile in patients with SUI and to replicate findings from a preceding study in which a significant difference in the urinary proteome was identified. Serum samples were collected from 38 patients (19 SUI; 19 matched, continent controls). Sample preparation included serum albumin depletion, in-solution enzymatic digestion of proteins applying a combination of Gluc-C and trypsin and peptide separation using nano High Performance Liquid Chromatography. Label-free quantitation of peptides and proteins was performed after triplicate measurements using quadrupole time-of-flight mass spectrometry. Peptide identification was achieved by searching the Human SwissProt Database using Mascot and X!Tandem. Main outcome measure was the relative abundance of each detected protein in serum. Of 7012 identified proteins, 33 proteins were induced (detected in SUI, not in controls) and five proteins were depleted (detected in controls, not in SUI). All depleted proteins play a role in immune/DNA damage response. Induced proteins are involved in inflammatory response, response to cellular stress, coagulation and cytoskeleton stability/ motility. Plasma serine protease inhibitor (SERPINA5) was found induced and previously also showed a higher abundance in urine samples of SUI patients. Data are available via ProteomeXchange with identifier PXD008553.

Folate Receptor ß Regulates Integrin CD11b/CD18 Adhesion of a Macrophage Subset to Collagen.

Folate, also known as vitamin B9, is necessary for essential cellular functions such as DNA synthesis, repair, and methylation. It is supplied to the cell via several transporters and receptors, including folate receptor (FR) ß, a GPI-anchored protein belonging to the folate receptor family. As FRß shows a restricted expression to cells of myeloid origin and only a subset of activated macrophages and placental cells have been shown to express functional FRß, it represents a promising target for future therapeutic strategies. In this study, we performed affinity purification and mass spectrometric analysis of the protein microenvironment of FRß in the plasma membrane of human FRß(+) macrophages and FRß-transduced monocytic THP-1 cells. In this manner, we identified a novel role of FRß: that is, we report functional interactions of FRß with receptors mediating cellular adhesion, in particular the CD11b/CD18 ß2 integrin heterodimer complement receptor type 3/Mac-1. This interaction results in impeded adhesion of FRß(+) human primary macrophages and THP-1 cells to collagen in comparison with their FRß(-) counterparts. We further show that FRß is only expressed by human macrophages when differentiated with M-CSF. These findings thus identify FRß as a novel CD11b/CD18 regulator for trafficking and homing of a subset of macrophages on collagen.

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression.

Immobilized monolithic enzymatic reactor and its application for analysis of in-vitro fertilization media samples.

Classical proteomics approaches involve enzymatic hydrolysis of proteins (either separated by polyacrylamide gels or in solution) followed by peptide identification using LC-MS/MS analysis. This method requires normally more than 16 h to complete. In the case of clinical analysis, it is of the utmost importance to provide fast and reproducible analysis with minimal manual sample handling. Herein we report the method development for online protein digestion on immobilized monolithic enzymatic reactors (IMER) to accelerate protein digestion, reduce manual sample handling, and provide reproducibility to the digestion process in clinical laboratory. An integrated online digestion and separation method using monolithic immobilized enzymatic reactor was developed and applied to digestion and separation of in-vitro-fertilization media.

Direct injection of HILIC fractions on the reversed-phase trap column improves protein identification rates for salivary proteins.

Online combination of hydrophilic interaction chromatography (HILIC) and RP chromatography for separation of tryptic peptides is a challenging approach due to the incompatibility of direct loading HILIC fractions on the RP trapping column. High amounts of organic modifiers in loading solvents decrease the binding efficiency of tryptic peptides on C18 phases and lower the number of identifications. A 500 µL loop upfront of the trapping column filled with aqueous mobile phase was employed as a mixing chamber and enabled direct injections and improved saliva protein identification rates of HILIC fractions.

Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies.

Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high-concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity-based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin-enriched fractions relative to the nondepleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively.

Urinary proteomic pattern in female stress urinary incontinence: a pilot study.

INTRODUCTION AND HYPOTHESIS: Previous studies aiming to identify specific pre-defined urine protein biomarkers for stress urinary incontinence (SUI) have not identified clinically important differences. The hypothesis of our study was that the global distribution of urinary proteins, the proteome, differs between women with and those without SUI. METHODS: In this age-matched case-control study, we compared the urinary proteome of 20 women with SUI and 20 controls. Proteins were identified by applying high-performance liquid chromatography separation and tandem mass spectrometry detection. Data analysis was performed using Mascot 2.4.1 embedded in ProteinScape 3.1. RESULTS: We identified 828 different proteins. The concentration of six of those showed a significant difference between urine samples of SUI patients and those of controls (q value < 0.25). Four proteins showed a higher abundance in SUI samples compared with controls: plasma serine protease inhibitor (logFC 1.11), leucine-rich alpha-2-glycoprotein (logFC 3.91), lysosomal alpha-glucosidase (logFC 1.24), and peptidyl-prolyl cis- trans isomerase A (logFC 1.96). We identified two proteins in lower abundance in SUI samples compared with controls: uromodulin (logFC -4.87) and TALPID3 (logFC -1.99). CONCLUSIONS: Overexpression of plasma serine protease inhibitor, leucine-rich alpha-2-glycoprotein, lysosomal alpha-glucosidase, and peptidyl-prolyl cis- trans isomerase A, and lower expression of uromodulin and TALPID3, in urine may be associated with female SUI.

KINFix--A formalin-free non-commercial fixative optimized for histological, immunohistochemical and molecular analyses of neurosurgical tissue specimens.

An optimal fixative should ideally combine the advantages of formalin fixation and freezing, allowing for good preservation of histology and molecular components, easy handling and storage, lack of toxicity, and low costs. Most of these criteria are fulfilled by ethanol-based solutions, and due to our good experience with the commercial RCL2 fixative, reflected by our published single-center trial, we initiated a multicenter ring trial. However, during its course, RCL2 was discontinued on the market. Therefore, we created our own agent, KINFix, composed of the same main constituents as RCL2, and employed it in our laboratory with similar results. Here we present our evaluation of the three fixatives formalin, RCL2, and KINFix from the perspective of histopathology as well as nucleic acid and protein analyses in comparison to fresh frozen tissues together with the multicenter ring trial data for RCL2. We observe that RCL2 and KINFix offer comparable histomorphology and superior template for molecular analyses than formalin. Moreover, KINFix as freely available fixative might overcome some of the difficulties related to the commercial agents. Therefore, we conclude that KINFix might be an attractive complement to formalin in tissue processing and advocate its use in neuropathological practice.

Differential Potential of Pharmacological PARP Inhibitors for Inhibiting Cell Proliferation and Inducing Apoptosis in Human Breast Cancer Cells.

BRCA1/2-mutant cells are hypersensitive to inactivation of poly(ADP-ribose) polymerase 1 (PARP-1). We recently showed that inhibition of PARP-1 by NU1025 is strongly cytotoxic for BRCA1-positive BT-20 cells, but not BRCA1-deficient SKBr-3 cells. These results raised the possibility that other PARP-1 inhibitors, particularly those tested in clinical trials, may be more efficacious against BRCA1-deficient SKBr-3 breast cancer cells than NU1025. Thus, in the presented study the cytotoxicity of four PARP inhibitors under clinical evaluation (olaparib, rucaparib, iniparib and AZD2461) was examined and compared to that of NU1025. The sensitivity of breast cancer cells to the PARP-1 inhibition strongly varied. Remarkably, BRCA-1-deficient SKBr-3 cells were almost completely insensitive to NU1025, olaparib and rucaparib, whereas BRCA1-expressing BT-20 cells were strongly affected by NU1025 even at low doses. In contrast, iniparib and AZD2461 were cytotoxic for both BT-20 and SKBr-3 cells. Of the four tested PARP-1 inhibitors only AZD2461 strongly affected cell cycle progression. Interestingly, the anti-proliferative and pro-apoptotic potential of the tested PARP-1 inhibitors clearly correlated with their capacity to damage DNA. Further analyses revealed that proteomic signatures of the two studied breast cancer cell lines strongly differ, and a set of 197 proteins was differentially expressed in NU1025-treated BT-20 cancer cells. These results indicate that BT-20 cells may harbor an unknown defect in DNA repair pathway(s) rendering them sensitive to PARP-1 inhibition. They also imply that therapeutic applicability of PARP-1 inhibitors is not limited to BRCA mutation carriers but can be extended to patients harboring deficiencies in other components of the pathway(s).

Proteome analysis of testis from infertile protein C inhibitor-deficient mice reveals novel changes in serpin processing and prostaglandin metabolism.

Serine protease inhibitors (serpin) have therapeutic potential in a variety of pathogenic processes, ranging from thrombosis and altered immune response to liver cirrhosis. To investigate the physiological effects of protein C inhibitor (PCI, serpinA5), its gene was inactivated in a mouse model, resulting in male infertility. In the present report, 2D differential gel electrophoresis was utilized to investigate the molecular mechanisms for PCI involvement in male reproduction. Comparing the testes proteomes of three PCI-knockout mice with three wild types demonstrated similar patterns with the exception of a massive upregulation of prostaglandin reductase 1 (tenfold; p < 0.002) and the complete shifts in the molecular weights of serpinA1C and serpinA3K. All these PCI-dependent proteome changes were immunologically verified. Unbiased proteome analysis indicated that inactivation of serpinA5 strongly influenced both the protein species pattern of other A-clade serpins as well as prostaglandin metabolism in the testes.

Empirical multidimensional space for scoring peptide spectrum matches in shotgun proteomics.

Data-dependent tandem mass spectrometry (MS/MS) is one of the main techniques for protein identification in shotgun proteomics. In a typical LC-MS/MS workflow, peptide product ion mass spectra (MS/MS spectra) are compared with those derived theoretically from a protein sequence database. Scoring of these matches results in peptide identifications. A set of peptide identifications is characterized by false discovery rate (FDR), which determines the fraction of false identifications in the set. The total number of peptides targeted for fragmentation is in the range of 10,000 to 20,000 for a several-hour LC-MS/MS run. Typically, <50% of these MS/MS spectra result in peptide-spectrum matches (PSMs). A small fraction of PSMs pass the preset FDR level (commonly 1%) giving a list of identified proteins, yet a large number of correct PSMs corresponding to the peptides originally present in the sample are left behind in the "grey area" below the identity threshold. Following the numerous efforts to recover these correct PSMs, here we investigate the utility of a scoring scheme based on the multiple PSM descriptors available from the experimental data. These descriptors include retention time, deviation between experimental and theoretical mass, number of missed cleavages upon in-solution protein digestion, precursor ion fraction (PIF), PSM count per sequence, potential modifications, median fragment mass error, (13)C isotope mass difference, charge states, and number of PSMs per protein. The proposed scheme utilizes a set of metrics obtained for the corresponding distributions of each of the descriptors. We found that the proposed PSM scoring algorithm differentiates equally or more efficiently between correct and incorrect identifications compared with existing postsearch validation approaches.

A platelet protein biochip rapidly detects an Alzheimer's disease-specific phenotype.

Alzheimer's disease (AD), a multifactorial neurodegenerative condition caused by genetic and environmental factors, is diagnosed using neuropsychological tests and brain imaging; molecular diagnostics are not routinely applied. Studies have identified AD-specific cerebrospinal fluid (CSF) biomarkers but sample collection requires invasive lumbar puncture. To identify AD-modulated proteins in easily accessible blood platelets, which share biochemical signatures with neurons, we compared platelet lysates from 62 AD, 24 amnestic mild cognitive impairment (aMCI), 13 vascular dementia (VaD), and 12 Parkinson's disease (PD) patients with those of 112 matched controls by fluorescence two-dimensional differential gel electrophoresis in independent discovery and verification sets. The optimal sum score of four mass spectrometry (MS)-identified proteins yielded a sensitivity of 94 % and a specificity of 89 % (AUC = 0.969, 95 % CI = 0.944-0.994) to differentiate AD patients from healthy controls. To bridge the gap between bench and bedside, we developed a high-throughput multiplex protein biochip with great potential for routine AD screening. For convenience and speed of application, this array combines loading control-assisted protein quantification of monoamine oxidase B and tropomyosin 1 with protein-based genotyping for single nucleotide polymorphisms (SNPs) in the apolipoprotein E and glutathione S-transferase omega 1 genes. Based on minimally invasive blood drawing, this innovative protein biochip enables identification of AD patients with an accuracy of 92 % in a single analytical step in less than 4 h.

A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection.

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.