Proteome of human endometrium: Identification of differentially expressed proteins in proliferative and secretory phase endometrium

Purpose: To exploit the potential of proteomics to identify and study additional yet‐unidentified important proteins present in human endometrium. Experimental design: The proteome of human endometrium would be established using 2‐DE and MALDI and the data analyzed to identify differential protein expression in the proliferative and secretory phase of the menstrual cycle using PDQuest software and MALDI. Results: In the present work, 2‐DE of human endometrium protein led to the resolution of over 200 spots. Subsequent MALDI analysis of 215 spots allowed the identification of 194 proteins. A total of 57 out of the 215 spots were found to be differentially expressed, out of which 49 could be identified using MALDI. These differentially expressed proteins included structural proteins, molecular chaperones, signaling proteins, metabolic proteins, proteins related to immunity, RNA biogenesis, protein biosynthesis and others. The differential expressions of seven representative proteins in secretory and proliferative phase endometrium tissue were confirmed by immunoblot analysis. Conclusion and clinical relevance: This study establishes the 2‐D proteome of human endometrium represented by 194 identified protein spots. The present data provides an important clue towards determining the function of these proteins with respect to endometrium related diseases.     

Proteomic expression analysis and comparison of protein and mRNA expression profiles in human malignant gliomas

Gliomas are highly heterogeneous and therapy resistant tumors with a poor prognosis. Novel experimental therapeutic approaches have shown some promising results, but often target specific molecular mechanisms or antigens, and careful characterization of the molecular subgroup of the tumors will therefore likely be important. Thorough investigations of gene and protein alterations are also important to better understand the tumorigenic mechanisms. We have undertaken a proteomic approach, using 2-D DIGE and LC-MS/MS protein identification, to investigate 38 human gliomas and normal brains. We show that the proteome profile can discriminate between normal brain and tumors, and between tumors of varying grade by a supervised classifier. Furthermore, an analysis of the identified proteins shows an enrichment of proteins associated to pathways known to be central in gliomas, such as MEK/Erk signaling and actin cytoskeleton. It also shows a shift between different glial fibrillary acidic protein (GFAP) representatives in different grades. In a previous study the gene expression profile was characterized in an almost identical set of tumors, which enabled a paired analysis of the gene and protein expression profiles. We show that there is often a weak correlation between the mRNA and protein level. This, together with the ability of proteomics to identify PTMs, emphasizes the benefit of characterization on a protein level.     

iTRAQ‐Based Quantitative Proteomic Analyses of High Grade Esophageal Squamous Intraepithelial Neoplasia

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and is the fourth most lethal cancer in China. Little is known about the proteome of high grade esophageal squamous intraepithelial neoplasia (HGN), which is a premalignant lesion of ESCC. A quantitative proteomic analysis using an isobaric tag for relative and absolute quantification (iTRAQ) approach is used to characterize the protein expression profiles in HGN. Among the 3156 identified proteins, a total of 236 proteins are discovered to be differentially expressed. Compared with paired normal esophageal epithelial tissues, 138 proteins are upregulated and 98 proteins are downregulated in HGN. Bioinformatics analyses are performed according to gene ontology, clusters of orthologous groups, and kyoto encyclopedia of genes and genomes enrichment analyses. Six differentially expressed proteins are chosen and validated by Western blotting. The results of the study increase our understanding of early tumorigenesis during ESCC, and provide insights into the proteome at the initial stages of the disease that can be used to identify potential biomarkers for early diagnosis and for therapeutic targets.     

Differential protein expression between normal, early-stage, and late-stage myxomatous mitral valves from dogs

Valvular heart disease accounts for over 20 000 deaths and 90 000 hospitalizations yearly in the United States. Myxomatous valve disease (MVD) is the most common disease of the mitral valve in humans and dogs. MVD is pathologically identical in these species and its pathogenesis is poorly understood. The objectives of this study were to (i) develop proteomic methodology suitable for analysis of extracellular matrix‐rich heart valve tissues and (ii) survey over‐ and under‐expressed proteins that could provide mechanistic clues into the pathogenesis of MVD. Normal, early‐stage, and late‐stage myxomatous mitral valves from dogs were studied. A shotgun proteomic analysis was used to quantify differential protein expression. Proteins were classified by function and clustered according to differential expression patterns. More than 300 proteins, with 117 of those being differentially expressed, were identified. Hierarchical sample clustering of differential protein profiles showed that early‐ and late‐stage valves were closely related. This finding suggests that proteome changes occur in early degeneration stages and these persist in late stages, characterizing a diseased proteome that is distinct from normal. Shotgun proteome analysis of matrix‐rich canine heart valves is feasible, and should be applicable to human heart valves. This study provides a basis for future investigations into the pathogenesis of MVD.     

A well-based reverse-phase protein array applicable to extracts from formalin-fixed paraffin-embedded tissue

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue offers significant diagnostic utility but is complicated due to the high level of covalently crosslinked proteins arising from formalin fixation. To address these challenges, we developed a reliable protein extraction method for FFPE tissue, based on heat-induced antigen retrieval within a pressure cooker. The protein extraction yield from archival FFPE tissue section is approximately 90% of that recovered from frozen tissue. This method demonstrates preservation of immunoreactivity and recovery of full-length proteins by Western blotting. Additionally, we developed a well-based RP protein array platform utilizing an electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, remain stable over 1?month at room temperature. Theses data suggest that this new protein-profiling platform coupled with the protein extraction method can be used for molecular profiling analysis in FFPE tissue, and contribute to the validation and development of biomarkers in clinical studies.     

Comprehensive profiling of metastasis-related proteins in paired hepatocellular carcinoma cells with different metastasis potentials

Precise and comprehensive identifications of the proteins associated with metastasis are critical for early diagnosis and therapeutic intervention of hepatocellular carcinoma (HCC). Therefore, we investigated the proteomic differences between a pair of HCC cell lines, originating from the same progenitor, with different metastasis potential using amino acid-coded mass tagging-based LC-MS/MS quantitative proteomic approach. Totally the relative abundance of 336 proteins in these cell lines were quantified, in which 121 proteins were upregulated by >30%, and 64 proteins were downregulated by >23% in the cells with high metastasis potential. Further validation studies by Western blotting in a series of HCC cell types with progressively increasing trend of metastasis showed that peroxiredoxin 4, HSP90β and HSP27 were positively correlated with increasing metastasis while prohibitin was negatively correlated with metastasis potential. These validation results were also consistent with that obtained from comparative analysis of clinic tissues samples. Function annotations of differentially expressed HCC proteome suggested that the emergence and development of high metastasis involved the dysregulation of cell migration, cell cycle and membrane traffics. Together our results revealed a much more comprehensive profile than that from 2-DE-based method and provided more global insights into the mechanisms of HCC metastasis and potential markers for clinical diagnosis.     

Guided protein extraction from formalin-fixed tissues for quantitative multiplex analysis avoids detrimental effects of histological stains

Formalin fixed and paraffin embedded (FFPE) tissues are the basis for histopathological diagnosis of many diseases around the world. For translational research and routine diagnostics, protein analysis from FFPE tissues is very important. We evaluated the potential influence of six histological stains, including hematoxylin (Mayer and Gill), fast red, light green, methyl blue and toluidine blue, for yield, electrophoretic mobility in 1-D gels, and immunoreactivity of proteins isolated from formalin-fixed breast cancer tissues. Proteins extracted from stained FFPE tissues using a recently established technique were compared with proteins obtained from the same tissues but without prior histological staining. Western blot and quantitative protein lysate microarray analysis demonstrated that histological staining can result in decreased protein yield but may not have much influence on immunoreactivity and electrophoretic mobility. Interestingly, not all staining protocols tested are compatible with subsequent protein analysis. The commonly used hematoxylin staining was found to be suitable for multiplexed quantitative protein measurement technologies although protein extraction was less efficient. For best results we suggest a guided protein extraction method, in which an adjacent hematoxylin/eosin-stained tissue section is used to control dissection of an unstained specimen for subsequent protein extraction and quantification for research and diagnosis.     

An evaluation of multidimensional fingerprinting in the context of clinical proteomics

Multidimensional fingerprinting (MDF) utilizes measurable peptide characteristics to identify proteins. In this study, 3‐D fingerprinting, namely, parent protein molecular weight, peptide mass, and peptide retention time on RPLC, is used to identify 331 differentially expressed proteins between normal and human colon cancer plasma membrane samples. A false discovery rate (FDR) procedure is introduced to evaluate the performance of MDF on the colon cancer dataset. This evaluation establishes a false protein identification rate below 15% for this dataset. Western blot analysis is performed to validate the differential expression of the MDF‐identified protein VDAC1 on the original tissue samples. The limits of MDF are further assessed by a simulation study where key parameters such as database size, query size, and mass accuracy are varied. The results of this simulation study demonstrate that fingerprinting with three dimensions yields low FDR values even for large queries on the complete human proteome without the need for prior peptide sequencing by tandem mass spectrometry. Specifically, when mass accuracy is 10 ppm or lower, full human proteome searches can achieve FDR values of 10% or less.     

Analysis of the differential proteome of human erythroblasts during in vitro erythropoiesis by 2-D DIGE

In the present study we have used an in vitro culture system that induces differentiation of human CD34(+) cells down the erythroid lineage along with 2-D DIGE to determine the differential proteome of erythroblasts at specific developmental stages during erythropoiesis. We initially distinguished 154 proteins differentially expressed between pro-normoblasts and polychromatic/orthochromatic erythroblasts, of which 24 protein spots, representing 21 different proteins, were identified following MS/MS and verification in replicate experiments with cells from different individuals. These data were confirmed by analysis of the differential proteome of erythroblasts at more discrete stages of erythropoiesis using 2-D DIGE and by mapping the expression of three identified proteins (Annexin I, Annexin II, Carbonic Anhydrase I) throughout erythropoiesis by Western blot with specific antisera. In addition, the differential expression of proteins due to biological variation, such as polymorphism, was determined by comparing erythroblasts at the same developmental stage from different individuals; none of the proteins thus identified were represented in the above data set. Finally, we discuss the problems associated with 2-D DIGE gel-based proteomic approaches such as ours and suggest a modified approach for decreased inter-gel variation, improved protein resolution and increased protein concentration, which should significantly facilitate protein identification.     

Discovery of putative oocyte quality markers by comparative ExacTag proteomics

Purpose: Identification of the biomarkers of oocyte quality, and developmental and reprogramming potential is of importance to assisted reproductive technology in humans and animals. Experimental design: PerkinElmer ExacTag? Kit was used to label differentially proteins in pig oocyte extracts (oocyte proteome) and pig oocyte‐conditioned in vitro maturation media (oocyte secretome) obtained with high‐ and low‐quality oocytes. Results: We identified 16 major proteins in the oocyte proteome that were expressed differentially in high‐ versus low‐quality oocytes. More abundant proteins in the high‐quality oocyte proteome included kelch‐like ECH‐associated protein 1 (an adaptor for ubiquitin‐ligase CUL3), nuclear export factor CRM1 and ataxia‐telangiectasia mutated protein kinase. Dystrophin (DMD) was more abundant in low‐quality oocytes. In the secretome, we identified 110 proteins, including DMD and cystic fibrosis transmembrane conductance regulator, two proteins implicated in muscular dystrophy and cystic fibrosis, respectively. Monoubiquitin was identified in the low‐quality‐oocyte secretome. Conclusions and clinical implications: A direct, quantitative proteomic analysis of small oocyte protein samples can identify potential markers of oocyte quality without the need for a large amount of total protein. This approach will be applied to discovery of non‐invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs.     

Proteomics is not only a biomarker discovery tool

The relatively young science of proteomics has been extensively used to identify biomarkers. However, a detailed and careful interpretation of proteomics data can also provide a clear picture of integrated biochemical systems, which can lead to a better comprehension of pathological processes. For example, the proteome analysis of human brain tissue from patients with schizophrenia, bipolar disorder, or multiple sclerosis compared with healthy controls has identified differentially expressed proteins that may not only be potential biomarkers but may also provide information that may increase the comprehension of these neurological disorders. Thus, proteomics is not only a biomarker discovery tool but can also identify potential players of relevance for diseases.     

An application of the 2-nitrobenzenesulfenyl method to proteomic profiling of human colorectal carcinoma: A novel approach for biomarker discovery

In the development of novel biomarkers, the proteomic approach is advantageous because using it the cancer-associated proteins can be directly identified. We previously developed a 2-nitrobenzenesulfenyl (NBS) method to improve quantitative proteome analysis. Here, we applied this method to proteomic profiling of colorectal carcinoma (CRC) to identify novel proteins with altered expression in CRC. Each pair of tumor and normal tissue specimens from 12 CRC patients was analyzed, and approximately 5000 NBS-labeled paired peaks were quantified. Peaks with altered signal intensities (>1.5-fold) and occurring frequently in the samples (>70%) were selected, and 128 proteins were identified by MS/MS analyses as differentially expressed proteins in CRC tissues. Many proteins were newly revealed to be CRC related; 30 were reported in earlier studies of CRC. Six proteins that were up-regulated in CRC (ZYX, RAN, RCN1, AHCY, LGALS1, and VIM) were further characterized and validated by Western blot and immunohistochemistry. All six were found to be CRC-localized, either in cancer cells or in stroma cells near the cancer cells. These results indicate that the proteins identified in this study are novel candidates for CRC markers, and that the NBS method is useful in proteome mining to discover novel biomarkers.     

Proteomics and the lung: Analysis of bronchoalveolar lavage fluid

Our knowledge of the complex bronchoalveolar lavage fluid (BALF) proteome has increased significantly over the last decade; but still, there remain many aspects of the BALF proteome that need characterization. Current proteomic methodologies resolve proteins within limited dynamic ranges: thereby, being limited in their ability to examine important areas of the BALF proteome, such as low molecular weight, low abundance proteins. To ensure proper coverage of these proteins in the BALF proteome, a refined 2-DE standard operation protocol is presented, highlighting important issues in sample collection, sample preparation, and 2-D DIGE analysis. It is hoped that this will help advance the field of BALF proteomics, BALFomics, which has lagged behind similar biofluids such as plasma and serum.     

Identification of glial-cell-line-derived neurotrophic factor-regulated proteins of striatum in mouse model of Parkinson disease

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for dopaminergic neurons, and hence serves as a therapeutic candidate for the treatment of Parkinson's disease. However, despite the potential clinical and physiological importance of GDNF, its mechanism of action is unclear. Therefore, we employed a state-of-the-art proteomic technique, DIGE, along with MS and a bioinformatics tool called Database for Annotation, Visualization and Integrated Discovery (DAVID), to profile proteome changes in the parkinsonian mouse striatum after GDNF challenge. Forty-six unique differentially expressed proteins were successfully identified, which were found either up-regulated and/or down-regulated at the two time points 4 and 72 h compared with the control. Proteins involved in cell differentiation and system development formed the largest part of the proteins regulated under GDNF. Furthermore, the aberrant expression of HSPs and mitochondria-associated proteins were noticeable. Moreover, mitochondrial stress 70 protein and heat shock cognate 71 kDa protein, whose relative levels increased significantly in GDNF-treated striatum, were further evaluated with Western blot and RT-PCR, demonstrating a good agreement with quantitative proteomic data. These data will provide some clues for understanding the mechanisms by which GDNF promotes the survival of dopaminergic neurons.     

Complete solubilization of formalin-fixed,paraffin-embedded tissue may improve proteomic studies

Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues.     

Quantitative proteomic analysis of a paired human liver healthy versus carcinoma cell lines with the same genetic background to identify potential hepatocellular carcinoma markers

To comprehensively measure global changes in protein expression associated with human hepatocellular carcinoma (HCC), comparative proteomic analysis of two cell lines derived from the healthy and carcinoma tissue of a same donor respectively was conducted using quantitative amino acid-coded mass tagging /stable isotope labeling with amino acids in cell culture-based LC-MS/MS approach. Among a total of 501 proteins precisely quantified, the expressions of 128 proteins were significantly altered including 70 proteins up-regulated and 58 down-regulated in HCC cells. According to their previously characterized functions, the differentially expressed proteins were found associated with nine functional categories including glycolysis, stress response, cell communication, cell cycle, apoptosis/death, etc. For example, multiple enzymes involving glycolysis pathway were found differentially regulated in HCC cells, illustrating the critical participation of glycolysis in the HCC transformation. The accuracy of certain differentially expressed proteins identified through the amino acid-coded mass tagging-based quantification was validated in the paired cell lines, and later their pathological correlations were examined in multiple clinical pairs of normal versus tumor tissues from HCC specimen by using a variety of biological approaches including Western blotting and in situ immunoassays. These consistencies suggested that multiple proteins such as HSP27, annexin V, glyceraldehyde-3-phosphate dehydrogenase, nucleolin and elongation factor Tu could be the biomarkers candidates for diagnosis of HCC.     

The untapped potential of proteomic analysis in pediatric pulmonary hypertension

Analysis of the human proteome has become increasingly sophisticated, and offers invaluable potential insight into the pathophysiology of human disease. The increasing standardization of methods, speed, and sophistication of mass spectrometric analysis, availability of reliable antibodies, and dissemination of information among the scientific community has allowed for exponential growth of our knowledge base. The continued effort to provide a molecular explanation for future medical applications based on biomarker discovery is epitomized by the outstanding efforts of the human proteome project, whose goal is to generate a map of the human proteome. However, proteomic analysis is underrepresented in pediatric illness; given the unique challenges of research in the pediatric population, proteomic analysis represents enormous untapped potential, especially in the further elucidation of the pathophysiology of rare diseases such as pulmonary hypertension (PH). In this article, we will describe the unique challenge of pediatric research, the importance of alternative avenues such as proteomics for in-depth analysis of pediatric pathobiology at the cellular level, the specific need for proteomic investigation of pediatric PH, the current status of PH proteomics, and future directions.