Proteomic Features Research Articles

Edible Cyanobacterial Genus Arthrospira: Actual State of the Art in Cultivation Methods, Genetics, and Application in Medicine.

The cyanobacterial genus Arthrospira appears very conserved and has been divided into five main genetic clusters on the basis of molecular taxonomy markers. Genetic studies of seven Arthrospira strains, including genome sequencing, have enabled a better understanding of those photosynthetic prokaryotes. Even though genetic manipulations have not yet been performed with success, many genomic and proteomic features such as stress adaptation, nitrogen fixation, or biofuel production have been characterized. Many of above-mentioned studies aimed to optimize the cultivation conditions. Factors like the light intensity and quality, the nitrogen source, or different modes of growth (auto-, hetero-, or mixotrophic) have been studied in detail. The scaling-up of the biomass production using photobioreactors, either closed or open, was also investigated to increase the production of useful compounds. The richness of nutrients contained in the genus Arthrospira can be used for promising applications in the biomedical domain. Ingredients such as the calcium spirulan, immulina, C-phycocyanin, and γ-linolenic acid (GLA) show a strong biological activity. Recently, its use in the fight against cancer cells was documented in many publications. The health-promoting action of “Spirulina” has been demonstrated in the case of cardiovascular diseases and age-related conditions. Some compounds also have potent immunomodulatory properties, promoting the growth of beneficial gut microflora, acting as antimicrobial and antiviral. Products derived from Arthrospira were shown to successfully replace biomaterial scaffolds in regenerative medicine. Supplementation with the cyanobacterium also improves the health of livestock and quality of the products of animal origin. They were also used in cosmetic preparations.

Blood-based biomarkers in lung cancer: prognosis and treatment decisions.

Despite recent advances, non-small cell lung cancer (NSCLC) remains a devastating disease with overall poor prognosis. Major contributing factors include obstacles to diagnosing the disease early in its course during the asymptomatic stage as well as diversity and complexity of its biology underlying tumorigenesis and tumor progression. Advances in molecularly targeted therapies which drives the development of personalized cancer care require precise and comprehensive understanding of tumor biology, not only at the time of diagnosis but also during treatment course and surveillance. As lung tumor tissue can be difficult to obtain without invasive and potentially risky procedures, it is difficult to monitor treatment response with serial tissue biopsies. Development of non-invasive but reliable blood based tumor markers has become an important research area. In this review, we focus on the following circulating biomarkers that have been identified in recent years: circulating tumor cells (CTCs); circulating cell-free nucleic acids, such as circulating tumor DNA (ctDNA) and microRNA (miR); and other biomarkers such as genomic and proteomic features. These biomarkers not only have prognostic values, but also can help guild treatment decisions by monitoring tumor burden, detecting minimal residual disease and/or recurrent disease, as well as monitoring evolution of genetic alterations throughout the treatment course.

Quantitative, in situ analysis of mRNAs and proteins with subcellular resolution

We describe here a method, termed immunoFISH, for simultaneous in situ analysis of the composition and distribution of proteins and individual RNA transcripts in single cells. Individual RNA molecules are labeled by hybridization and target proteins are concurrently stained using immunofluorescence. Multicolor fluorescence images are acquired and analyzed to determine the abundance, composition, and distribution of hybridized probes and immunofluorescence. We assessed the ability of immunoFISH to simultaneous quantify protein and transcript levels and distribution in cultured HER2 positive breast cancer cells and human breast tumor samples. We demonstrated the utility of this assay in several applications including demonstration of the existence of a layer of normal myoepithelial KRT14 expressing cells that separate HER2+ cancer cells from the stromal and immune microenvironment in HER2+ invasive breast cancer. Our studies show that immunoFISH provides quantitative information about the spatial heterogeneity in transcriptional and proteomic features that exist between and within cells.

Transcriptomic and proteomic feature of salt stress-regulated network in Jerusalem artichoke (Helianthus tuberosus L.) root based on de novo assembly sequencing analysis.

Ribosome activation and sugar metabolic process mainly act on the regulation of salt tolerance in the bioenergy crop Helianthus tuberosus L. as dissected by integrated transcriptomic and proteomic analyses. Helianthus tuberosus L. is an important halophyte plant that can survive in saline-alkali soil. It is vitally necessary to build an available genomic resource to investigate the molecular mechanisms underlying salt tolerance in H. tuberosus. De novo assembly and annotation of transcriptomes were built for H. tuberosus using a HiSeq4000 platform. 293,823 transcripts were identified and annotated into 190,567 unigenes. In addition, iTRAQ-labeled quantitative proteomics was carried out to detect global protein profiling as a response to salt stress. Comparative omics analysis showed that 5432 genes and 43 proteins were differentially expressed in H. tuberosus under salt stress, which were enriched in the following processes: carbohydrate metabolism, ribosome activation and translation, oxidation-reduction and ion binding. The reprogramming of transcript and protein works suggested that the induced activity of ribosome and sugar signaling may endue H. tuberosus with salt tolerance. With high-quality sequencing and annotation, the obtained transcriptomics and proteomics provide a robust genomic resource for dissecting the regulatory molecular mechanism of H. tuberosus in response to salt stress.

Collagen VI Null Mice as a Model for Early Onset Muscle Decline in Aging.

Collagen VI is an extracellular matrix (ECM) protein playing a key role in skeletal muscles and whose deficiency leads to connective tissue diseases in humans and in animal models. However, most studies have been focused on skeletal muscle features. We performed an extensive proteomic profiling in two skeletal muscles (diaphragm and gastrocnemius) of wild-type and collagen VI null (Col6a1−/−) mice at different ages, from 6- (adult) to 12- (aged) month-old to 24 (old) month-old. While in wild-type animals the number of proteins and the level of modification occurring during aging were comparable in the two analyzed muscles, Col6a1−/− mice displayed a number of muscle-type specific variations. In particular, gastrocnemius displayed a limited number of dysregulated proteins in adult mice, while in aged muscles the modifications were more pronounced in terms of number and level. In diaphragm, the differences displayed by 6-month-old Col6a1−/− mice were more pronounced compared to wild-type mice and persisted at 12 months of age. In adult Col6a1−/− mice, the major variations were found in the enzymes belonging to the glycolytic pathway and the tricarboxylic acid (TCA) cycle, as well as in autophagy-related proteins. When compared to wild-type animals Col6a1−/− mice displayed a general metabolic rewiring which was particularly prominent the diaphragm at 6 months of age. Comparison of the proteomic features and the molecular analysis of metabolic and autophagic pathways in adult and aged Col6a1−/− diaphragm indicated that the effects of aging, culminating in lipotoxicity and autophagic impairment, were already present at 6 months of age. Conversely, the effects of aging in Col6a1−/− gastrocnemius were similar but delayed becoming apparent at 12 months of age. A similar metabolic rewiring and autophagic impairment was found in the diaphragm of 24-month-old wild-type mice, confirming that fatty acid synthase (FASN) increment and decreased microtubule-associated proteins 1A/1B light chain 3B (LC3B) lipidation are hallmarks of the aging process. Altogether these data indicate that the diaphragm of Col6a1−/− animal model can be considered as a model of early skeletal muscle aging.

Native DIGE: Efficient Tool to Elucidate Protein Interactomes.

Protein-protein interactions and multi-protein assemblies are inherent features of proteomes, involving soluble and membrane proteins. This imparts structural and functional heterogeneity to the proteome. One needs to consider this aspect while studying changes in abundance or activities of proteins in response to any physiological stimulus. Abundance changes in components of a given proteome can be best visualized and quantified using electrophoresis-based approaches. Here, we describe the method of Blue Native Difference Gel Electrophoresis (BN DIGE) to quantify abundance changes in proteins in the context of protein-protein interactions. This method confers an additional advantage to monitor quantitative changes in membrane proteins, which otherwise is a difficult task.

Serum Proteomic Variability Associated with Clinical Phenotype in Familial Transthyretin Amyloidosis (ATTRm).

Transthyretin (TTR), normally a plasma circulating protein, can become misfolded and aggregated, ultimately leading to extracellular deposition of amyloid fibrils usually targeted to heart or nerve tissues. Referred to as TTR-associated amyloidoses (ATTR), this group of diseases is frequently life threatening and fatal if untreated. ATTR, caused by amyloid-forming variant TTR proteins (ATTRm) that arise from point mutations in the TTR gene, were classically referred to as familial amyloid cardiomyopathy (FAC) or familial amyloid polyneuropathy (FAP), reflecting the clinical phenotype. FAC and FAP are pathologies that can be challenging to diagnose as there are no definitive biomarkers of disease; moreover, disease-specific measures of progression are lacking, and treatment options are limited. Thus, the discovery of sensitive and specific indicators of disease has the potential to improve recognition, enable accurate measurement of amyloid progression and response to treatment, and reveal key information regarding FAC and FAP pathobiological mechanisms. In this study, the goal was to investigate serum proteomic features unique to FAC and FAP types of ATTRm. Multiple-reaction monitoring mass spectrometry (MRM-MS), a powerful technique in profiling proteomes, was used to measure the serum concentrations of 160 proteins in samples from FAC and FAP patients. Results were compared to data from healthy control sera obtained from individuals matched to age (≥60 years), gender (male), and race (Caucasian). Proteomic analyses of ATTRm (FAC and FAP) and control samples showed significant concentration differences in 107 of 192 (56%) of the serum proteins that were studied. In comparing FAC to FAP, differences in concentrations as well as interactions and functions of several proteins were identified as unique to each disease; significantly lower levels of TTR were specific to FAC, but not to FAP. Annotated functional clustering identified extracellular region, signal, and signal peptide as terms common to FAC and FAP. Conversely, disulfide bond was unique to FAC; secreted, glycosylation site: N-linked, glycosylation, glycoprotein, polymorphism, and sequence variant were associated solely with FAP. Predicted protein-protein associations in FAC were seen for reaction, binding, and activation processes; no associations were found in FAP. This study demonstrates significant proteomic differences between ATTRm patient and control sera, as well as ATTRm phenotype-associated variations in the circulating levels of several proteins including TTR. The identification of serum proteins unique to FAC and FAP may have diagnostic and prognostic utility and could possibly provide important clues about disease mechanisms.

Proteomic Features of Colorectal Cancer Identify Tumor Subtypes Independent of Oncogenic Mutations and Independently Predict Relapse-Free Survival.

The directed study of the functional proteome in colorectal cancer (CRC) has identified critical protein markers and signaling pathways; however, the prognostic relevance of many of these proteins remains unclear. We determined the prognostic implications of the functional proteome in 263 CRC tumor samples from patients treated at MD Anderson Cancer Center (MDACC) and 462 patients from The Cancer Genome Atlas (TCGA) to identify patterns of protein expression that drive tumorigenesis. A total of 163 validated proteins were analyzed by reverse phase protein array (RPPA). Unsupervised hierarchical clustering of the tumor proteins from the MDACC cohort was performed, and clustering was validated using RPPA data from TCGA CRC. Cox regression was used to identify predictors of tumor recurrence. Clustering revealed dichotomization, with subtype A notable for a high epithelial-mesenchymal transition (EMT) protein signature, while subtype B was notable for high Akt/TSC/mTOR pathway components. Survival data were only available for the MDACC cohort and were used to evaluate prognostic relevance of these protein signatures. Group B demonstrated worse relapse-free survival (hazard ratio 2.11, 95% confidence interval 1.04-4.27, p=0.039), although there was no difference in known genomic drivers between the two proteomic groups. Proteomic grouping and stage were significant predictors of recurrence on multivariate analysis. Eight proteins were found to be significant predictors of tumor recurrence on multivariate analysis: Collagen VI, FOXO3a, INPP4B, LcK, phospho-PEA15, phospho-PRAS40, Rad51, phospho-S6. CRC can be classified into distinct subtypes by proteomic features independent of common oncogenic driver mutations. Proteomic analysis has identified key biomarkers with prognostic importance, however these findings require further validation in an independent cohort.

P.241 - Comparative high resolution proteomic analysis of dystrophic mouse models reveals a core dystrophic proteome and the impact of aging

The most common form of childhood muscular dystrophy is Duchenne muscular dystrophy (DMD), which is a severe, X-linked muscle wasting disorder. The disease is caused by mutations in the DMD gene, leading to loss of the dystrophin protein which in turn causes progressive muscle degeneration and premature death. Various mouse models have been developed to study the disease, such as the commonly used mdx mouse, which has a nonsense mutation in exon 23, though this mutation is not characteristically present in humans. Another model is the mdx52 mouse, which has an exon 52 deletion in the mutation “hotspot” region of the DMD gene. To better understand the dystrophic phenotype and its progression with aging, we undertook for the first-time comparative proteomic analysis of both mdx and mdx52 models, using high-resolution isoelectric Focusing Liquid Chromatography-tandem Mass Spectrometry (HiRIEF-LC-MS/MS) with Tandem Mass Tag (TMT) isobaric labelling of tibialis anterior (TA) skeletal muscle to identify proteomic characteristics at 8, 16 and 80 weeks of age. Comparative proteomic analysis of mdx, mdx52 and C57BL/6 control mice identified 4,974 proteins in 27plex format and revealed a core set of proteomic changes reflective of the dystrophic phenotype. Proteomic analysis with age identified core proteomic features of normal muscle aging, overlap of these with progression of the dystrophic phenotype and features specific to the dystrophic phenotype with age. This study provides novel insight into specific proteomic changes characteristic of muscle aging and the relationship of these to dystrophic pathology over time.

Proteomic features of delayed ocular symptoms caused by exposure to sulfur mustard: As studied by protein profiling of corneal epithelium

Exposure to mustard gas can lead to variations in the proteome of corneal epithelium cells and after a latency period produces delayed symptoms in the eyes of chemical victims. Hence, a comparative proteome analysis was conducted between the corneal epithelial cells of chemical victims from Iran-Iraq war (1980–1988) and healthy donors. To this end, corneal epithelium samples from victims and healthy individuals were collected, and the proteome of these samples were prepared for two-dimensional electrophoresis and the analysis of spots by statistical software. Selected spots were further analyzed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Twenty four proteins were identified of which eighteen proteins showed downregulation while six proteins were upregulated in the victims in comparison to the normal individuals. Also, six protein spots were confirmed by western-blot analysis. In conclusion, all the twenty-four identified proteins are involved in pathways which their up- or down-regulation leads to the accumulation of undesired substrates, cell death and apoptosis. Bioinformatics' tools indicated that these identified proteins were involved in various metabolic processes, DNA damage response, immune response and etc. The present study provides a suitable platform for further clinical studies.

Identification of Proteomic Features To Distinguish Benign Pulmonary Nodules from Lung Adenocarcinoma.

We hypothesized that distinct protein expression features of benign and malignant pulmonary nodules may reveal novel candidate biomarkers for the early detection of lung cancer. We performed proteome profiling by liquid chromatography-tandem mass spectrometry to characterize 34 resected benign lung nodules, 24 untreated lung adenocarcinomas (ADCs), and biopsies of bronchial epithelium. Group comparisons identified 65 proteins that differentiate nodules from ADCs and normal bronchial epithelium and 66 proteins that differentiate ADCs from nodules and normal bronchial epithelium. We developed a multiplexed parallel reaction monitoring (PRM) assay to quantify a subset of 43 of these candidate biomarkers in an independent cohort of 20 benign nodules, 21 ADCs, and 20 normal bronchial biopsies. PRM analyses confirmed significant nodule-specific abundance of 10 proteins including ALOX5, ALOX5AP, CCL19, CILP1, COL5A2, ITGB2, ITGAX, PTPRE, S100A12, and SLC2A3 and significant ADC-specific abundance of CEACAM6, CRABP2, LAD1, PLOD2, and TMEM110-MUSTN1. Immunohistochemistry analyses for seven selected proteins performed on an independent set of tissue microarrays confirmed nodule-specific expression of ALOX5, ALOX5AP, ITGAX, and SLC2A3 and cancer-specific expression of CEACAM6. These studies illustrate the value of global and targeted proteomics in a systematic process to identify and qualify candidate biomarkers for noninvasive molecular diagnosis of lung cancer.

A Classification Model to Predict the Rate of Decline of Kidney Function.

The African American Study of Kidney Disease and Hypertension (AASK), a randomized double-blinded treatment trial, was motivated by the high rate of hypertension-related renal disease in the African-American population and the scarcity of effective therapies. This study describes a pattern-based classification approach to predict the rate of decline of kidney function using surface-enhanced laser desorption ionization/time of flight proteomic data from rapid and slow progressors classified by rate of change in glomerular filtration rate. An accurate classification model consisting of 7 out of 5,751 serum proteomic features is constructed by applying the logical analysis of data (LAD) methodology. On cross-validation by 10-folding, the model was shown to have an accuracy of 80.6 ± 0.11%, sensitivity of 78.4 ± 0.17%, and specificity of 78.5 ± 0.16%. The LAD discriminant is used to identify the patients in different risk groups. The LAD risk scores assigned to 116 AASK patients generated a receiver operating curves curve with AUC 0.899 (CI 0.845–0.953) and outperforms the risk scores assigned by proteinuria, one of the best predictors of chronic kidney disease progression.

Abstract 2204: A proteomic landscape of diffuse-type gastric cancer

Abstract Gastric cancer is a heterogeneous disease characterized by poor clinical outcomes and limited targeted treatment options. Among them, diffuse-type gastric cancer (DGC) is the subtype with worst prognosis. Here we describe the first proteomic landscape of DGC. We carried out proteome profiling and targeted exome DNA sequencing of 84 DGC samples. We analyzed the 1,008 (168 x 6) raw files together for uniformed quality control and protein identification with 1% global protein false discovery rate (FDR), which resulted in the identification of 11,340 gene products (GPs). A SAM (significance analysis of microarray) analysis identified 1,641 proteins as differentially expressed between T (tumor) and N (nearby) with statistical significance (FDR q value<0.01 by SAM and differential expression ratio>0.5/ < -0.5), including 1,211 up-regulated and 430 down-regulated GPs. Gene Ontology annotation indicated that tumor proteomes were significantly enriched in cell cycle, DNA replication, checkpoint, E2F, WNT, p53 signaling, epithelial mesenchymal transition (EMT), and inflammation/cytokine-receptor interaction pathways, and the proteomes of the nearby tissues are enriched in metabolism pathways, such as fatty acid metabolism, oxidative phosphorylation, and amino acid metabolism. Notably, many gastric makers (ANXA10, VSIG1, CLDN18, CTSE, TFF2, MUC5AC and MUC6) and signature proteins for stomach functions, including digestion, absorption, secretion, and stomach acid generation (PGC, GIF, GAST, and ATP4A), were lost in tumors. Based on proteome profiling alone, DGC can be subtyped into 3 major classes (PX1-3) that exhibit distinct proteome features and correlate with distinct clinical outcomes (Gehan-Breslow-Wilcoxon P = 0.024). PX1 exhibits proteome stability and the best overall survival; PX2 exhibits dysregulation in DNA replication and cell cycle, and is most sensitive to chemotherapy; PX3 features hyper-activated immune response and is not responsive to chemotherapy. We identified seven-marker proteins that can stratify DGC patients into these three subtypes, opening a door for proteome subtyping in clinical application and intervention. Furthermore, we nominated drug target candidates taking into consideration both the altered DGC proteome and association data with patients’ overall survival. This study revealed the altered signaling pathways in DGC and demonstrated the advantage of proteomic approach in molecular subtyping of cancer. Citation Format: Sai Ge, Xia Xia, Chen Ding, Bei Zhen, Quan Zhou, Jinwen Feng, Jiajia Yuan, Rui Chen, Yumei Li, Zhongqi Ge, Jiafu Ji, Lianhai Zhang, Jiayuan Wang, Zhongwu Li, Yumei Lai, Ying Hu, Yanyan Li, Yilin Li, Jing Gao, Lin Chen, Jianming Xu, Chunchao Zhang, Sung Yun Jung, Mingwei Liu, Lei Song, Wanlin Liu, Gaigai Guo, Tongqing Gong, Yin Huang, Yang Qiu, Tieliu Shi, Weimin Zhu, Yi Wang, Fuchu He, Lin Shen, Jun Qin, CNHPP. A proteomic landscape of diffuse-type gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2204. doi:10.1158/1538-7445.AM2017-2204

Genomic, proteomic and bioinformatic analysis of two temperate phages in Roseobacter clade bacteria isolated from the deep-sea water

BackgroundMarine phages are spectacularly diverse in nature. Dozens of roseophages infecting members of Roseobacter clade bacteria were isolated and characterized, exhibiting a very high degree of genetic diversity. In the present study, the induction of two temperate bacteriophages, namely, vB_ThpS-P1 and vB_PeaS-P1, was performed in Roseobacter clade bacteria isolated from the deep-sea water, Thiobacimonas profunda JLT2016 and Pelagibaca abyssi JLT2014, respectively. Two novel phages in morphological, genomic and proteomic features were presented, and their phylogeny and evolutionary relationships were explored by bioinformatic analysis.ResultsElectron microscopy showed that the morphology of the two phages were similar to that of siphoviruses. Genome sequencing indicated that the two phages were similar in size, organization, and content, thereby suggesting that these shared a common ancestor. Despite the presence of Mu-like phage head genes, the phages are more closely related to Rhodobacter phage RC1 than Mu phages in terms of gene content and sequence similarity. Based on comparative genomic and phylogenetic analysis, we propose a Mu-like head phage group to allow for the inclusion of Mu-like phages and two newly phages. The sequences of the Mu-like head phage group were widespread, occurring in each investigated metagenomes. Furthermore, the horizontal exchange of genetic material within the Mu-like head phage group might have involved a gene that was associated with phage phenotypic characteristics.ConclusionsThis study is the first report on the complete genome sequences of temperate phages that infect deep-sea roseobacters, belonging to the Mu-like head phage group. The Mu-like head phage group might represent a small but ubiquitous fraction of marine viral diversity.

Quantitative Proteomics Analysis Confirmed Oxidative Metabolism Predominates inStreptomyces coelicolorversus Glycolytic Metabolism inStreptomyces lividans

Recent physiological studies indicated that S. lividans metabolism was mainly glycolytic, whereas S. coelicolor metabolism was mainly oxidative. To determine whether such metabolic characteristics were correlated with consistent proteomics features, a comparative label-free, shotgun proteomics analysis of these strains was carried out. Among 2024 proteins identified, 360 showed significant differences in abundance between the strains. This study revealed that S. coelicolor catabolized glucose less actively than S. lividans, whereas the amino acids present in the medium were catabolized less actively by S. lividans than by S. coelicolor. The abundance of glycolytic proteins in S. lividans was consistent with its high glycolytic activity, whereas the abundance of proteins involved in the catabolism of amino acids in S. coelicolor provided an explanatory basis for its predominantly oxidative metabolism. In this study, conducted under conditions of low O2 availability, proteins involved in resistance to oxidative stress and those belonging to a DosR-like dormancy regulon were abundant in S. coelicolor, whereas tellurium resistance proteins were abundant in S. lividans. This indicated that the strains reacted differently to O2 limitation. Proteins belonging to the CDA, RED, and ACT pathways, usually highly expressed in S. coelicolor, were not detected under these conditions, whereas proteins of siderophores, 5-hydroxyectoine, and terpenoid biosynthetic pathways were present.

Proteomic features linked to tenderness of aged pork loins.

There is considerable evidence that the protein component of fresh pork makes a major contribution to tenderness. In particular, the proteomic profile can be linked to postmortem events including pH decline, tissue oxidation, and protein degradation. The objectives for this study were to determine differences in sarcoplasmic proteomes that contribute to tenderness variation in aged pork longissimus dorsi muscles (LM). A defined set of pork loins selected to be similar in pH, color, and lipid yet different in tenderness were used. Pork loins were assigned to tenderness groups based on their star probe values; a high star probe group (HSP; n=12 mean star probe 7.75 kg) and low star probe group (LPS; n=12 star probe 4.95 kg) Samples were selected for proteomic experiments based on star probe values, and selected samples were within specified ranges for ultimate pH (5.54-5.86), marbling score (1.0-3.0), and percent total lipid (1.61-3.37%). Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry were used to examine sarcoplasmic protein abundance and potential modifications. Proteins spots that were significantly different across groups were selected for identification. Results from 2D-DIGE showed that HSP samples had significantly more abundant metabolic, stress response, and regulatory proteins in the sarcoplasmic fraction compared with LSP samples. The stress response protein peroxiredoxin-2 was more abundant in HSP samples as determined by 2D-DIGE ( ≤ 0.01; 2 spots) and western blot assay ( = 0.02). Low star probe samples showed significantly more degradation of the structural protein desmin in 2D-DIGE ( < 0.01) and western blot assay ( < 0.01). These results demonstrate that extreme proteolytic differences influenced measured tenderness of LSP and HSP samples and that soluble desmin and peroxiredoxin-2 may be used as biomarkers to differentiate between tough and tender aged pork products.

Proteomics for the Investigation of Mycobacteria

The physiology of Mycobacterium tuberculosis, the causative agent of tuberculosis, is being studied with intensity. However, despite the genomic and transcriptomic data available today, the pathogenic potential of these bacteria remains poorly understood. Therefore, proteomic approaches seem relevant in studying mycobacteria. This review covers the main stages in the proteomic analysis methods used to study mycobacteria. The main achievements in the area of M. tuberculosis proteomics are described in general. Special attention is paid to the proteomic features of the Beijing family, which is widespread in Russia. Considering that the proteome is a set of all the proteins in the cell, post-translational modifications of mycobacterium proteins are also described.

Proteomic Characteristics of Blood Serum in Rats with Different Behavioral Parameters after Acute Stress.

We studied proteome profile of blood serum of Wistar rats with different behavioral activity immediately and in 1 and 3 days after acute stress on the model of 12-h immobilization during the nighttime. Comparative analysis of 2D-electrophoretograms revealed differences in the expression of serum proteins in non-stressed (control) and stressed (experimental) rats. We found 22 protein spots that characterized the proteomic features of blood serum in rats with different prognostic resistance to stress. Mass-spectrometry of isolated spots identified 6 functional proteins. Persistent proteome changes in the blood of animals at different stages after acute stress were determined. The specificity of proteomic characteristics of blood serum was shown in behaviorally passive and active rats during the post-stress period. These data extend the concept on specific protein markers for the formation of a negative emotional state and adaptive-and-compensatory processes in mammals with different sensitivity to stressogenic factors.

Integrated Molecular Characterization of Uterine Carcinosarcoma

We performed genomic, epigenomic, transcriptomic, and proteomic characterizations of uterine carcinosarcomas (UCSs). Cohort samples had extensive copy-number alterations and highly recurrent somatic mutations. Frequent mutations were found in TP53, PTEN, PIK3CA, PPP2R1A, FBXW7, and KRAS, similar to endometrioid and serous uterine carcinomas. Transcriptome sequencing identified a strong epithelial-to-mesenchymal transition (EMT) gene signature in a subset of cases that was attributable to epigenetic alterations at microRNA promoters. The range of EMT scores in UCS was the largest among all tumor types studied via The Cancer Genome Atlas. UCSs shared proteomic features with gynecologic carcinomas and sarcomas with intermediate EMT features. Multiple somatic mutations and copy-number alterations in genes that are therapeutic targets were identified.

Abstract P1-01-13: A new workflow comprising size based CTC enrichment followed by in situ labeling and micromanipulation with CellCelector™ enables label-free enrichment, isolation and characterization of circulating tumor cells in breast cancer

Abstract Circulating tumor cells (CTCs) are believed to be metastatic precursors of most solid epithelial tumors. Therefore, characterization of their genomic, transcriptomic, and proteomic features may improve cancer diagnosis and prognosis as well as treatment selection and monitoring. CTCs are mostly detected by systems utilizing expression of the epithelial cell adhesion molecule (EpCAM) on CTCs. However, CTCs which have acquired mesenchymal-like features are overlooked although they are believed to be the most malignant cells. Moreover, due to inter cellular heterogeneity single cell isolation by e.g. micromanipulation is required to achieve cellular resolution. In this project we established a workflow combining the Parsortix™ system to enrich for EpCAMlow/negative CTCs by size and deformability and the CellCelector™ micromanipulator to singularize them for further molecular characterizations. First, our workflow was validated by cell-line spiking experiments into blood from healthy subjects (MCF7). Captured cells were stained in situ for nuclei, cytokeratin and leukocyte markers, were harvested and then micromanipulated as single cells reaching a capturing rate of 62.66% (± 17.79) and a harvesting rate of 72% (± 29.61). Then this workflow was applied to a cohort of metastatic breast cancer blood samples which were previously depleted for EpCAMhigh cells. In 18 out of 22 samples 541 EpCAMhigh CTCs and in 16 samples 257 EpCAMlow/negative potential CTCs were found. Of these 156 EpCAMlow/negative and comparable numbers of EpCAMhigh cells were successfully isolated and lysed for whole genome amplification (Ampli1™). Successful genome-wide amplification was observed in 6.5% of EpCAMlow/negative cells vs. 30% of EpCAMhigh cells indicating lower quality of genomic DNA in the first subpopulation. Sanger sequencing of PCR-amplified exons 9 and 20 of PIK3CA gene revealed heterogeneous mutation status in EpCAMlow/negative cells from the same patient. High quality WGA products were also processed for array-based comparative genomic hybridization (aCGH). Comparison of resulting profiles to 267 archived aCGH profiles from breast cancer single cells identified similar somatic copy number alterations: i.e. chromosome 1p loss and 1q gain confirming the tumor origin of captured EpCAMlow/negative cells. In addition we could also identify some different aberrations which need to be further investigated. In summary, our workflow combining size based CTC enrichment followed by in situ labeling and micromanipulation is suitable for single tumor cell analysis. Citation Format: Neubauer HJ, Lampignano R, Neumann MHD, Behrens B, Franken A, Stoecklein N, Niederacher D, Fehm TN. A new workflow comprising size based CTC enrichment followed by in situ labeling and micromanipulation with CellCelector™ enables label-free enrichment, isolation and characterization of circulating tumor cells in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-13.