Proteomic analysis of maternal serum in down syndrome: identification of novel protein biomarkers

Down syndrome (DS) is the most prevalent chromosomal disorder, accounting for significant morbidity and mortality. Definitive diagnosis requires invasive amniocentesis, and current maternal serum-based testing requires a false-positive rate of about 5% to detect 85% of affected pregnancies. We have performed a comprehensive proteomic analysis to identify potential serum biomarkers to detect DS. First- and second-trimester maternal serum samples of DS and gestational age-matched controls were analyzed using multiple, complementary proteomic approaches, including fluorescence 2-dimensional gel electrophoresis (2D-DIGE), 2-dimensional liquid chromatography-chromatofocusing (2D-CF), multidimensional protein identification technology (MudPIT; LC/LC-MS/MS), and MALDI-TOF-MS peptide profiling. In total, 28 and 26 proteins were differentially present in first- and second-trimester samples, respectively. Of these, 19 were specific for the first trimester and 16 for the second trimester, and 10 were differentially present in both trimesters. Analysis of MALDI-TOF-MS peptide profiles with pattern-recognition software also discriminated between DS and controls in both trimesters, with an average recognition capability approaching 96%. A majority of the biomarkers identified are serum glycoproteins that may play a role in cellular differentiation and growth of fetus. Further characterization and quantification of these markers in a larger cohort of subjects may provide the basis for new tests for improved DS screening.     

Proteomic analysis of cervical-vaginal fluid: identification of novel biomarkers for detection of intra-amniotic infection

Intra-amniotic infection (IAI) is associated with preterm birth and perinatal mortality. To identify potential biomarkers, we performed a comprehensive survey of the cervical-vaginal fluid (CVF) proteome from a primate IAI model utilizing multidimensional protein identification technology (LC/LC-MS/MS) and MALDI-TOF-MS analyses. Analyses of CVF proteome identified 205 unique proteins and differential expression of 27 proteins in controls and IAI samples. Protein expression signatures and immunodetection of specific biomarkers identified can be employed for noninvasive detection of IAI.     

Quantitative proteome and acidic subproteome profiling of Candida albicans yeast-to-hypha transition

Candida albicans yeast-to-hypha morphological transition is involved in the virulence strategy of this opportunistic fungal pathogen. Changes in relative abundance of the Candida proteome related to this process were analyzed using different two-dimensional differential in-gel electrophoresis (2D-DIGE)-based approaches. First, a comparative analysis of yeast and hyphal cytoplasmic proteins allowed the detection of 106 protein spots with significant variation in abundance. Sixty-one of them, corresponding to 46 proteins, were identified. As most of the differentially abundant proteins had an acidic isoelectric point, a large-scale prefractionation approach to analyze the acidic C. albicans subproteome was carried out. Ninety acidic C. albicans proteins were identified by either gel-based or nongel-based approaches. Additionally, different workflows combining preparative isoelectric focusing, Cy labeling, and narrow pH gradient 2-DE gels were tested to analyze the differences in relative protein abundance between yeast and hyphal acidic subproteomes. It was possible to identify 21 differentially abundant acidic proteins; 10 of them were not identified in the previous 2D-DIGE gels. Functional and network interaction analyses of the 56 differentially abundant proteins identified by both approaches rendered an integrated view of metabolic and cellular process reorganization during the yeast-to-hypha transition. With these results, we propose a model of metabolic reorganization.     

Identification of bactenecin-1 in cervicovaginal fluid by two-dimensional electrophoresis in an ovine model of preterm labour

Preterm labour is a major problem in obstetrics. Timely intervention with available treatments is hampered by the lack of a reliable test of imminent preterm delivery. Current methods of diagnosis are based on the detection of breakdown products of foetal membranes or structural changes to the cervix when preterm labour is well established. The aim of this study was to screen the cervicovaginal fluid (CVF) proteome to identify labour-associated proteins that could be used as markers of imminent preterm delivery. Labour was induced in sheep at 135 days of gestation (term 147 days) by foetal infusion of dexamethasone (1 mg/24 h). CVF samples were collected before and 28 h after the start of infusion as well as at delivery (58.7 +/- 1.9 after the start of infusion, n = 5). One protein that was upregulated eight-fold, was bactenecin-1, a member of the cathelicidin family of antimicrobial proteins. This antimicrobial protein warrants further investigation as a marker of preterm labour, particularly during the period after the initiation of labour but before there is marked cervical connective tissue breakdown.     

Proteomics of breast carcinoma

Beast cancer is the most diagnosed cancer in women, accounting for approximately 40,000 deaths annually in the USA. Significant advances have been made in the areas of detection and treatment, but a significant number of breast cancers are detected late. The advent of proteomics provides the hope of discovering novel biological markers that can be used for early detection, disease diagnosis, prognostication and prediction of response to therapy. Several proteomics technologies including 2D-PAGE, 2D-DIGE, ICAT, SELDI-TOF, MudPIT and protein arrays have been used to uncover molecular mechanisms associated with breast carcinoma at the global level, and a number of these technologies, particularly the SELDI-TOF hold promise as a proteomic approach that can be applied at the bedside for discovering protein patterns that distinguish disease and disease-free states with high sensitivity and specificity. Laser microdissection, a method for selection of homogenous cell populations, coupled to 2D-DIGE or MudPIT constitute a new proteomics-based paradigm for detecting disease in pathology specimens and monitoring disease response to therapy. This review describes proteomics technologies, and their application in the proteomic analysis of breast carcinoma.     

Placenta proteome analysis from Down syndrome pregnancies for biomarker discovery

Down syndrome is one of the most frequent chromosomal disorders, with a prevalence of approximately 1/500 to 1/800, depending on the maternal age distribution of the pregnant population. However, few reliable protein biomarkers have been used in the diagnosis of this disease. Recent progress in quantitative proteomics has offered opportunities to discover biomarkers for tracking the progression and for understanding the molecular mechanisms of Down syndrome. In the present study, placental samples were analyzed by fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 101 proteins have been firmly identified representing 80 unique gene products. These proteins mainly function in cytoskeleton structure and regulation (such as vimentin and Profilin-1). Additionally, our quantitative proteomics approach has identified numerous previously reported Down syndrome markers, such as myelin protein. Here we present several Down syndrome biomarkers including galectin-1, ataxin-3 and sprouty-related EVH1 domain-containing protein 2 (SPRED2), which have not been reported elsewhere and may be associated with the progression and development of the disease. In summary, we report a comprehensive placenta-based proteomics approach for the identification of potential biomarkers for Down syndrome, in which serum amyloid P-component (APCS) and ataxin-3 have been shown to be up-regulated in the maternal peripheral plasma of Down syndrome cases. The potential of utilizing these markers for the prognosis and screening of Down syndrome warrants further investigation.     

Serum Protein Signatures Differentiating Autoimmune Pancreatitis versus Pancreatic Cancer

Autoimmune pancreatitis (AIP) is defined by characteristic lymphoplasmacytic infiltrate, ductal strictures and a pancreatic enlargement or mass that can mimic pancreatic cancer (PaCa). The distinction between this benign disease and pancreatic cancer can be challenging. However, an accurate diagnosis may pre-empt the misdiagnosis of cancer, allowing the appropriate medical treatment of AIP and, consequently, decreasing the number of unnecessary pancreatic resections.Mass spectrometry (MS) and two-dimensional differential gel electrophoresis (2D-DIGE) have been applied to analyse serum protein alterations associated with AIP and PaCa, and to identify protein signatures indicative of the diseases. Patients'' sera were immunodepleted from the 20 most prominent serum proteins prior to further 2D-DIGE and image analysis. The identity of the most-discriminatory proteins detected, was performed by MS and ELISAs were applied to confirm their expression. Serum profiling data analysis with 2D-DIGE revealed 39 protein peaks able to discriminate between AIP and PaCa. Proteins were purified and further analysed by MALDI-TOF-MS. Peptide mass fingerprinting led to identification of eleven proteins. Among them apolipoprotein A-I, apolipoprotein A-II, transthyretin, and tetranectin were identified and found as 3.0-, 3.5-, 2-, and 1.6-fold decreased in PaCa sera, respectively, whereas haptoglobin and apolipoprotein E were found to be 3.8- and 1.6-fold elevated in PaCa sera. With the exception of haptoglobin the ELISA results of the identified proteins confirmed the 2D-DIGE image analysis characteristics. Integration of the identified serum proteins as AIP markers may have considerable potential to provide additional information for the diagnosis of AIP to choose the appropriate treatment.     

A Potential Novel Spontaneous Preterm Birth Gene,AR, Identified by Linkage and Association Analysis of X Chromosomal Markers

Preterm birth is the major cause of neonatal mortality and morbidity. In many cases, it has severe life-long consequences for the health and neurological development of the newborn child. More than 50% of all preterm births are spontaneous, and currently there is no effective prevention. Several studies suggest that genetic factors play a role in spontaneous preterm birth (SPTB). However, its genetic background is insufficiently characterized. The aim of the present study was to perform a linkage analysis of X chromosomal markers in SPTB in large northern Finnish families with recurrent SPTBs. We found a significant linkage signal (HLOD  = 3.72) on chromosome locus Xq13.1 when the studied phenotype was being born preterm. There were no significant linkage signals when the studied phenotype was giving preterm deliveries. Two functional candidate genes, those encoding the androgen receptor (AR) and the interleukin-2 receptor gamma subunit (IL2RG), located near this locus were analyzed as candidates for SPTB in subsequent case-control association analyses. Nine single-nucleotide polymorphisms (SNPs) within these genes and an AR exon-1 CAG repeat, which was previously demonstrated to be functionally significant, were analyzed in mothers with preterm delivery (n = 272) and their offspring (n = 269), and in mothers with exclusively term deliveries (n = 201) and their offspring (n = 199), all originating from northern Finland. A replication study population consisting of individuals born preterm (n = 111) and term (n = 197) from southern Finland was also analyzed. Long AR CAG repeats (≥26) were overrepresented and short repeats (≤19) underrepresented in individuals born preterm compared to those born at term. Thus, our linkage and association results emphasize the role of the fetal genome in genetic predisposition to SPTB and implicate AR as a potential novel fetal susceptibility gene for SPTB.     

Mapping a new spontaneous preterm birth susceptibility gene, IGF1R, using linkage, haplotype sharing, and association analysis

Preterm birth is the major cause of neonatal death and serious morbidity. Most preterm births are due to spontaneous onset of labor without a known cause or effective prevention. Both maternal and fetal genomes influence the predisposition to spontaneous preterm birth (SPTB), but the susceptibility loci remain to be defined. We utilized a combination of unique population structures, family-based linkage analysis, and subsequent case-control association to identify a susceptibility haplotype for SPTB. Clinically well-characterized SPTB families from northern Finland, a subisolate founded by a relatively small founder population that has subsequently experienced a number of bottlenecks, were selected for the initial discovery sample. Genome-wide linkage analysis using a high-density single-nucleotide polymorphism (SNP) array in seven large northern Finnish non-consanginous families identified a locus on 15q26.3 (HLOD 4.68). This region contains the IGF1R gene, which encodes the type 1 insulin-like growth factor receptor IGF-1R. Haplotype segregation analysis revealed that a 55 kb 12-SNP core segment within the IGF1R gene was shared identical-by-state (IBS) in five families. A follow-up case-control study in an independent sample representing the more general Finnish population showed an association of a 6-SNP IGF1R haplotype with SPTB in the fetuses, providing further evidence for IGF1R as a SPTB predisposition gene (frequency in cases versus controls 0.11 versus 0.05, P = 0.001, odds ratio 2.3). This study demonstrates the identification of a predisposing, low-frequency haplotype in a multifactorial trait using a well-characterized population and a combination of family and case-control designs. Our findings support the identification of the novel susceptibility gene IGF1R for predisposition by the fetal genome to being born preterm.     

Extracellular vesicle RNAs reflect placenta dysfunction and are a biomarker source for preterm labour

Preterm birth (PTB) can lead to lifelong complications and challenges. Identifying and monitoring molecular signals in easily accessible biological samples that can diagnose or predict the risk of preterm labour (PTL) in pregnant women will reduce or prevent PTBs. A number of studies identified putative biomarkers for PTL including protein, miRNA and hormones from various body fluids. However, biomarkers identified from these studies usually lack consistency and reproducibility. Extracellular vesicles (EVs) in circulation have gained significant interest in recent years as these vesicles may be involved in cell‐cell communication. We have used an improved small RNA library construction protocol and a newly developed size exclusion chromatography (SEC)‐based EV purification method to gain a comprehensive view of circulating RNA in plasma and its distribution by analysing RNAs in whole plasma and EV‐associated and EV‐depleted plasma. We identified a number of miRNAs in EVs that can be used as biomarkers for PTL, and these miRNAs may reflect the pathological changes of the placenta during the development of PTL. To our knowledge, this is the first study to report a comprehensive picture of circulating RNA, including RNA in whole plasma, EV and EV‐depleted plasma, in PTL and reveal the usefulness of EV‐associated RNAs in disease diagnosis.     

Proteomic identification of plasma biomarkers in uterine leiomyoma

Recent progresses in quantitative proteomics have offered opportunities to discover plasma proteins as biomarkers for tracking the progression and for understanding the molecular mechanisms of uterine leiomyomas. In the present study, plasma samples were analyzed by fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 20 proteins have been firmly identified representing 13 unique gene products. These proteins mainly functioned in transportation (such as apolipoprotein A-I) and coagulation (such as fibrinogen gamma chain). Additionally, our quantitative proteomic approach has identified numerous previous reported plasma markers of uterine leiomyomas such as alpha-1-antitrypsin. On the contrary, we have presented several putative uterine leiomyomas biomarkers including afamin, apolipoprotein A-I, carbonic anhydrase 1, fibrinogen beta chain, fibrinogen gamma chain, gelsolin, hemopexin, leucine-rich alpha-2-glycoprotein, serotransferrin and vitamin D-binding protein which have not been reported and may be associated with the progression and development of the disease. In summary, we report a comprehensive patient-based proteomic approach for the identification of potential plasma biomarkers for uterine leiomyomas. The potential of utilizing these markers for screening and treating uterine leiomyomas warrants further investigations.     

LCM纯化的鼻咽癌间质和正常鼻咽间质的定量蛋白质组学研究

间质在肿瘤发生发展中的作用越来越受到重视．为寻找与鼻咽癌( nasopharyngeal carcinoma,NPC )发生发展相关的特异性间质蛋白,采用激光捕获显微切割技术( laser capture microdissection,LCM )纯化鼻咽癌间质和正常鼻咽黏膜间质,荧光差异双向凝胶电泳( fluorescent two-dimensional difference gel electrophoresis 2-D,DIGE )结合质谱技术分离鉴定间质相关蛋白．Western blot及免疫组织化学技术验证了其中3个差异蛋白(CapG、L-plastin和S100A9),证实了2D-DIGE结果的可靠性．建立了LCM 纯化的鼻咽癌间质和正常鼻咽间质的荧光差异蛋白表达图谱,高通量筛选与肿瘤发生相关的间质蛋白,共得到34个有统计学意义的蛋白质点,质谱鉴定得到20个差异蛋白．研究结果提示：这些差异表达的蛋白质将有助于阐明鼻咽癌细胞和周围间质的关系．对间质蛋白功能的进一步研究,将有助于解析间质在肿瘤发生中的作用机制,并为从间质途径寻找肿瘤治疗靶标提供新思路．     

Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis

In this study, we identified the differentially expressed proteins of female and male adults of Angiostrongylus cantonensis through differential proteomics. We extracted and purified total proteins from male and female adults, separated proteins by two-dimensional difference gel electrophoresis (2D-DIGE) in pH 4-7, analyzed the gel images by DeCyder 7.0 software, and sacrificed the infected rats to count the number of male and female adults. It was found 28 protein spots that were differentially expressed; seven protein spots were then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five proteins were up-regulated and two proteins down-regulated in male adults compared with female adults. Three of the five up-regulated proteins with known functions ascribed to them were identified as galectin-1, proteasome alpha subunit and peroxiredoxin. The two down-regulated proteins were identified as indoleamine dioxygenase like-myoglobin and galectin. Furthermore, the female was significantly greater than male adults (P<0.01) in the rats. The findings demonstrate the differences in protein expression profiles and the ability to survive in the final host between female and male adults of A. cantonensis, and may provide a theoretical basis to study their developmental biology further.     

Comprehensive proteomic analysis of human cervical-vaginal fluid

Cervical-vaginal fluid (CVF) is a potential rich source of biomarkers for enhancing our understanding of human parturition and pathologic conditions affecting pregnancy. In this study, we performed a comprehensive survey of the CVF proteome in pregnancy utilizing multidimensional liquid chromatography (2D-LC) coupled with mass spectrometry and gel-electrophoresis-based protein separation and identification. In total, 150 unique proteins were identified using multiple protein identification algorithms. Metabolism (32%) and immune response-related (22%) proteins are the major functional categories represented in the CVF proteome. A comparison of the CVF, serum, and amniotic fluid proteomes showed that 77 proteins are unique to CVF, while 56 and 17 CVF proteins also occur in serum and amniotic fluid, respectively. This data set provides a foundation for evaluation of these proteins as potential CVF biomarkers for noninvasive diagnosis of pregnancy-related disorders.     

Comparative 2D-DIGE Proteomic Analysis of Bovine Mammary Epithelial Cells during Lactation Reveals Protein Signatures for Lactation Persistency and Milk Yield

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.     

Two dimensional difference gel electrophoresis analysis of cerebrospinal fluid in tuberculous meningitis patients

Tuberculous meningitis (TBM) is a serious complication of tuberculosis that affects the central nervous system. Present methods to diagnose TBM are not suitable for early diagnosis. Molecular markers and sensitive methods to identify them in the early stage of infection of TBM are critically needed for efficient management. We have done the proteomic analysis of TBM cerebrospinal fluid (n=20) with 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 11 human proteins and 8 mycobacterial proteins with changed expression levels in comparison to controls. Arachidonate 5-lipoxygenase and glial fibrillary acidic protein, two of the identified proteins, were validated with western blot technique on a larger set of disease and control samples (n=40). These two proteins were also analyzed in fungal meningitis samples. We suggest that arachidonate 5-lipoxygenase can be considered for validation as a potential marker for diagnosis of TBM.     

Identification of Cellular Calcium Binding Protein Calmodulin as a Regulator of Rotavirus A Infection during Comparative Proteomic Study

Rotavirus (RV) being the major diarrhoegenic virus causes around 527000 children death (<5years age) worldwide. In cellular environment, viruses constantly adapt and modulate to survive and replicate while the host cell also responds to combat the situation and this results in the differential regulation of cellular proteins. To identify the virus induced differential expression of proteins, 2D-DIGE (Two-dimensional Difference Gel Electrophoresis) based proteomics was used. For this, HT-29 cells were infected with RV strain SA11 for 0 hours, 3 hours and 9 hours post infection (hpi), differentially expressed spots were excised from the gel and identified using MALDI-TOF/TOF mass spectrometry. 2D-DIGE based proteomics study identified 32 differentially modulated proteins, of which 22 were unique. Some of these were validated in HT-29 cell line and in BALB/c mice model. One of the modulated cellular proteins, calmodulin (CaM) was found to directly interact with RV protein VP6 in the presence of Ca²⁺. Ca²⁺-CaM/VP6 interaction positively regulates RV propagation since both CaM inhibitor (W-7) and Ca²⁺ chelator (BAPTA-AM) resulted in decreased viral titers. This study not only identifies differentially modulated cellular proteins upon infection with rotavirus in 2D-DIGE but also confirmed positive engagement of cellular Ca²⁺/CaM during viral pathogenesis.     

The effect of selenium enrichment on baker's yeast proteome

The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE)-tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p ≤ 0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially was verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY.     

Worker honeybee brain proteome

A large-scale mapping of the worker honeybee brain proteome was achieved by MudPIT. We identified 2742 proteins from forager and nurse honeybee brain samples; 17% of the total proteins were found to be differentially expressed by spectral count sampling statistics and a G-test. Sequences were compared with the EuKaryotic Orthologous Groups (KOG) catalog set using BLASTX and then categorized into the major KOG categories of most similar sequences. According to this categorization, nurse brain showed increased expression of proteins implicated in translation, ribosomal structure, and biogenesis (14.5%) compared with forager (1.8%). Experienced foragers overexpressed proteins involved in energy production and conversion, showing an extensive difference in this set of proteins (17%) in relation to the nurse subcaste (0.6%). Examples of proteins selectively expressed in each subcaste were analyzed. A comparison between these MudPIT experiments and previous 2-DE experiments revealed nine coincident proteins differentially expressed in both methodologies.