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The Epstein-Barr virus (EBV)-encoded oncoprotein latent membrane protein 1 (LMP1) constitutively activates nuclear factor κB (NFκB) from intracellular membranes to promote cell growth and survival. LMP1 associates with CD63 in intracellular membranes and is released via exosomes. Whether tumour necrosis factor (TNF) receptor-associated factors (TRAFs) mediate LMP1 NFκB signalling from endosomes and modulate exosomal sorting is unknown. In this article, we show that LMP1-TRAF2 signalling complexes accumulate at endosomes in a palmitoylation-dependent manner, thereby driving LMP1-dependent oncogenicity. Palmitoylation is a reversible post-translational modification and is considered to function as a membrane anchor for proteins. Mutagenesis studies showed that LMP1-TRAF2 trafficking to endosomes is dependent on one single cysteine residue (C78), a known palmitoylation site of LMP1. Notably, growth assays in soft agar revealed that oncogenic properties of the palmitoylation-deficient LMP1 mutant C78A were diminished compared to wild-type LMP1. Since LMP1 recruitment of TRAF2 and downstream NFκB signalling were not affected by a disturbance in palmitoylation, the specific localization of LMP1 at endosomal membranes appears crucial for its transforming potential. The importance of palmitoylation for trafficking to and signalling from endosomal membranes was not restricted to LMP1, as similar observations were made for the cellular oncoproteins Src and Fyn. Despite abundant LMP1-TRAF2 association at endosomal membranes TRAF2 could not be detected in exosomes by Western blotting or proteomics. Interestingly, point mutations that prevented TRAF binding strongly promoted the sorting and release of LMP1 via exosomes. These observations reveal that LMP1-TRAF2 complexes at endosomes support oncogenic NFκB activation and suggest that LMP1 dissociates from the activated signalling complexes upon sorting into intraluminal vesicles. We propose that "signalling endosomes" in EBV-infected tumour cells can fuse with the plasma membrane, explaining LMP1 release via exosomes.

miR-200a has been implicated in the pathogenesis of meningiomas, one of the most common central nervous system tumors in humans. To identify how miR-200a contributes to meningioma pathogenesis at the molecular level, we used a comparative protein profiling approach using Gel-nanoLC-MS/MS and identified approximately 130 dysregulated proteins in miR-200a-overexpressing meningioma cells. Following the bioinformatic analysis to identify potential genes targeted by miR-200a, we focused on the non-muscle heavy chain IIb (NMHCIIb), and showed that miR-200a directly targeted NMHCIIb. Considering the key roles of NMHCIIb in cell division and cell migration, we aimed to identify whether miR-200a regulated these processes through NMHCIIb. We found that NMHCIIb overexpression partially rescued miR-200a-mediated inhibition of cell migration, as well as cell growth in vitro and in vivo. Moreover, siRNA-mediated silencing of NMHCIIb expression resulted in a similar migration phenotype in these cells and inhibited meningioma tumor growth in mice. Taken together, these results suggest that NMHCIIb might serve as a novel therapeutic target in meningiomas.

The need for effective treatments halting Alzheimer's disease (AD) urges the discovery of the earliest possible biomarkers. Agrin is increased in the early stages of AD and is involved in amyloid-β (Aβ) fibrillation and synaptogenesis. We investigated the potential of agrin as an early AD cerebrospinal fluid (CSF) biomarker. We analyzed the agrin CSF concentration in nondemented controls (n = 20) and those with mild (n = 20) and severe (n = 20) AD. The levels of agrin CSF were not significantly divergent among the different patient groups and did not correlate with the concentration of Aβ42, total tau, phosphorylated tau, or the Mini Mental State Examination scores. However, agrin strongly correlated with age in those with dementia. The results indicate that agrin cannot be used as an early AD CSF biomarker using the current immunoassay. However, our population was relatively young; thus, the correlation between agrin and age suggests that stronger differences in agrin concentrations might be found in older groups with more heterogeneous AD pathologic features.

In order to evaluate whether ovarian volume, presence and diameter of the corpus luteum (CL) have effects on the number and quality of bovine recovered oocytes, 110 ovaries were obtained from the slaughterhouse. Cumulus oocytes complex were aspirated and evaluated under stereomicroscope. Oocytes were counted and classified according to their quality (Grades I, II, III and IV). Ovarian volume was weakly correlated to the number of good quality oocytes (P < 0.05). Ovaries with CL showed greater numbers of good quality oocytes than ovaries without CL (P < 0.05). Further, presence of CL and its diameter positively influenced the probability of recovering good quality oocytes (P < 0.05). In conclusion, ovarian volume is not a good parameter itself to predict important ovarian characteristics; moreover, analysis of CL, its presence and diameter, may be a good tool to improve efficiency on in vitro embryo production programs., (© 2014 Japanese Society of Animal Science.)

Background: Extracellular vesicles (EVs) are small nanometre-sized vesicles that are circulating in blood. They are released by multiple cells, including tumour cells. We hypothesized that circulating EVs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors., Methods: EVs released by U87 glioma cells, H3255 and H1650 non-small-cell lung cancer (NSCLC) cells were profiled by tandem mass spectrometry. Total AKT/protein kinase B and extracellular signal regulated kinase 1/2 (ERK1/2) levels as well as their relative phosphorylation were measured by western blot in isogenic U87 cells with or without mutant epidermal growth factor receptor (EGFRvIII) and their corresponding EVs. To assess biomarker potential, plasma samples from 24 healthy volunteers and 42 patients with cancer were used., Results: In total, 130 different protein kinases were found to be released in EVs including multiple drug targets, such as mammalian target of rapamycin (mTOR), AKT, ERK1/2, AXL and EGFR. Overexpression of EGFRvIII in U87 cells results in increased phosphorylation of EGFR, AKT and ERK1/2 in cells and EVs, whereas a decreased phosphorylation was noted upon treatment with the EGFR inhibitor erlotinib. EV samples derived from patients with cancer contained significantly more protein (p=0.0067) compared to healthy donors. Phosphorylation of AKT and ERK1/2 in plasma EVs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells. Preliminary analysis of total AKT and ERK1/2 levels in plasma EVs from patients with NSCLC before and after sorafenib/metformin treatment (n=12) shows a significant decrease in AKT levels among patients with a favourable treatment response (p<0.005)., Conclusion: Phosphorylation of protein kinases in EVs reflects their phosphorylation in tumour cells. Total AKT protein levels may allow monitoring of kinase inhibitor responses in patients with cancer.

Colorectal tumorigenesis is accompanied by the generation of oxidative stress, but how this controls tumor development is poorly understood. Here, we studied how the H2O2-reducing enzyme glutathione peroxidase 2 (GPx2) regulates H2O2 stress and differentiation in patient-derived "colonosphere" cultures. GPx2 silencing caused accumulation of radical oxygen species, sensitization to H2O2-induced apoptosis, and strongly reduced clone- and metastasis-forming capacity. Neutralization of radical oxygen species restored clonogenic capacity. Surprisingly, GPx2-suppressed cells also lacked differentiation potential and formed slow-growing undifferentiated tumors. GPx2 overexpression stimulated multilineage differentiation, proliferation, and tumor growth without reducing the tumor-initiating capacity. Finally, GPx2 expression was inversely correlated with H2O2-stress signatures in human colon tumor cohorts, but positively correlated with differentiation and proliferation. Moreover, high GPx2 expression was associated with early tumor recurrence, particularly in the recently identified aggressive subtype of human colon cancer. We conclude that H2O2 neutralization by GPx2 is essential for maintaining clonogenic and metastatic capacity, but also for the generation of differentiated proliferating tumor mass. The results reveal an unexpected redox-controlled link between tumor mass formation and metastatic capacity., (©2014 American Association for Cancer Research.)

BRI2 protein binds amyloid precursor protein to halt amyloid-β production and inhibits amyloid-β aggregation via its BRICHOS-domain suggesting a link between BRI2 and Alzheimer's disease (AD). Here, we investigate the possible involvement of BRI2 in human AD pathogenesis. BRI2 containing BRICHOS-domain was increased up to 3-fold in AD hippocampus (p = 0.003, n = 14/group). Immunohistochemistry showed BRI2 deposits associated with amyloid-β plaques in early pathologic stages (Braak-III; Thal-2/3). We observed a decrease in the protein levels of ADAM10 (p = 0.02) and furin (p = 0.066), as well as an increase in SPPL2b (p < 0.0001) in AD hippocampus. Because these enzymes are involved in BRI2 processing, their changes may lead to aberrant processing of BRI2 promoting its deposition and likely affecting BRI2 function. Loss of BRI2 function in AD was supported by the decreased presence of BRI2-amyloid precursor protein complexes in the hippocampus of AD patients compared with control subjects. In conclusion, our data obtained from human samples indicate that in early stages of AD there is an increased deposition of BRI2, which likely leads to impaired BRI2 function thereby influencing AD pathophysiology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) is a major health problem. Biomarkers associated with molecular changes in cancer cells can aid early detection, diagnosis, prognosis, therapy selection, and disease monitoring. Tumor tissue secretomes are a rich source of candidate biomarkers. To identify CRC protein biomarkers, secretomes of four pairs of human CRC tissue and patient-matched normal colon tissue samples, and secretomes of five CRC cell lines were analyzed by GeLC-MS/MS. Subsequent data analysis was based on label-free spectral counting, Ingenuity Pathway Analysis, Secretome/SignalP, STRING and Cytoscape, resulting in 2703 protein identifications in the tissue secretomes, of which 409 proteins were significantly more present in CRC samples than in controls. Biomarker selection of 76 candidates was based on consistent and abundant over-representation in cancer- compared to control-secretomes, and presumed neoplastic origin. Overlap analysis with previously obtained datasets revealed 21 biomarkers suited for early detection of CRC. Immunohistochemistry confirmed overexpression in CRC of one candidate marker (MCM5). In conclusion, a human reference dataset of 76 candidate biomarkers was identified for which we illustrate that combination with existing pre-clinical datasets allows pre-selection of biomarkers for blood- or stool-based assays to support clinical management of CRC. Further dedicated validation studies are required to demonstrate their clinical applicability., Biological Significance: Tissue secretome proteomes are a rich source of candidate biomarkers. Several secretome proteome datasets have been obtained from pre-clinical in vitro and in vivo colorectal cancer (CRC) model systems, yielding promising CRC biomarkers obtained under well-defined experimentally controlled conditions. However, which of these biomarker proteins are actually secreted by human CRC samples was not known. To our knowledge, this is the first study that directly compares secretome proteomes from clinically relevant human CRC tissues to patient-matched normal colon tissues. We identified 76 human CRC protein biomarkers that may facilitate blood-based or stool-based assay development to support clinical management of CRC. Overlap analysis with datasets from well-defined pre-clinical studies helps to determine what clinical application suits these human CRC biomarkers best, i.e. early detection, diagnosis, prognosis, therapy selection, and/or disease monitoring of CRC. This is demonstrated for a CRC mouse model dataset, revealing 21 human CRC biomarkers suited for early detection of CRC., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Breast cancer is traditionally considered as a heterogeneous disease. Molecular profiling of breast cancer by gene expression studies has provided us an important tool to discriminate a number of subtypes. These breast cancer subtypes have been shown to be associated with clinical outcome and treatment response. In order to elucidate the functional consequences of altered gene expressions related to each breast cancer subtype, proteomic technologies can provide further insight by identifying quantitative differences at the protein level. In recent years, proteomic technologies have matured to an extent that they can provide proteome-wide expressions in different clinical materials. This technology can be applied for the identification of proteins or protein profiles to further refine breast cancer subtypes or for discovery of novel protein biomarkers pointing towards metastatic potential or therapy resistance in a specific subtype. In this review, we summarize the current state of knowledge of proteomic research on molecular breast cancer classification and discuss important aspects of the potential usefulness of proteomics for discovery of breast cancer-associated protein biomarkers in the clinic., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Proteomics is optimally suited to bridge the gap between genomic information on the one hand and biologic functions and disease phenotypes at the other, since it studies the expression and/or post-translational modification (especially phosphorylation) of proteins--the major cellular players bringing about cellular functions--at a global level in biologic specimens. Mass spectrometry technology and (bio)informatic tools have matured to the extent that they can provide high-throughput, comprehensive, and quantitative protein inventories of cells, tissues, and biofluids in clinical samples at low level. In this article, we focus on next-generation proteomics employing nanoliquid chromatography coupled to high-resolution tandem mass spectrometry for in-depth (phospho)protein profiling of tumor tissues and (proximal) biofluids, with a focus on studies employing clinical material. In addition, we highlight emerging proteogenomic approaches for the identification of tumor-specific protein variants, and targeted multiplex mass spectrometry strategies for large-scale biomarker validation. Below we provide a discussion of recent progress, some research highlights, and challenges that remain for clinical translation of proteomic discoveries.

The aim of this study was to evaluate the effects of vitamin E associated with rapid thawing on cryopreserved goat semen. Two bucks were used and eight ejaculates per animal were collected using artificial vagina. Semen was diluted with the following treatments: BIOXCELL (control), BIOXCELL + Equex (sodium lauryl sulphate) and BIOXCELL + vitamin E 100 μM. Semen was packaged into 0.25 mL straws and cooled at 5°C for 1 hour. Freezing was performed in liquid nitrogen vapor (-155°C) during 15 minutes. Then, the straws were immersed in liquid nitrogen (-196°C). Straws were thawed at 38°C/60 seconds or at 60°C/7 seconds with immediate sperm analysis. Hypoosmotic swelling test was performed adding a 20 μL aliquot of thawed semen to 1 mL of hypoosmotic solution (100 mOsm · Kg(-1)) followed by incubation during 60 minutes in water bath (38°C). Vitamin E did not affect any studied parameters (P > 0.05). Nevertheless, defrosting rate of 60°C/7 seconds improved sperm membrane functional integrity (P < 0.05). Current knowledge about goat semen cryopreservation is not sufficient to ensure high post-thawing recovery rates; thus, this study brings important data about using antioxidants and different thawing rates on cryopreservation process.

Background: Cancer cells are able to change the protein expression and behavior of non-cancerous surrounding cells. Exosomes, secreted by prostate cancer (PCa) cells, may have a functional role in cancer metastasis and present a promising source for protein biomarkers. The aim of the present study was to identify which proteins in exosomes can influence non-cancerous cells, and to determine whether we can use urine exosomal proteins to identify high-risk PCa patients., Method: Exosomes were isolated by ultracentrifugation. Migration and invasion were studied by the transwell (invasion) assay. Proteomics was performed by LC-MS/MS and identified proteins were validated by Western blotting. Cellular uptake of fluorescent labeled PKH67-exosomes was measured by FACS., Results: Based on comparative protein profiling by mass spectrometry-based proteomics of LNCaP- and PC3-exosomes, we selected ITGA3 and ITGB1, involved in migration/invasion, for further analyses. Inhibition of exosomal ITGA3 reduced the migration and invasion of non-cancerous prostate epithelial cells (prEC) almost completely. Cellular uptake of exosomes by prEC was higher with PC3-exosomes compared to LNCaP exosomes. Finally, ITGA3 and ITGB1 were more abundant in urine exosomes of metastatic patients (p<0.05), compared to benign prostate hyperplasia or PCa., Conclusion: These data indicate exosomal ITGA3 and ITGB1 may play a role in manipulating non-cancerous surrounding cells and that measurement of ITGA3 and ITGB1 in urine exosomes has the potential to identify patients with metastatic PCa in a non-invasive manner.

Drug-resistant cancer stem cells (CSCs) have been implicated in tumor recurrence following chemotherapy. However, the contribution of CSCs to drug-resistance in colorectal cancer is unclear and CSC-intrinsic drug-resistance mechanisms are ill-defined. Here, we address these issues by proteomic analysis of the secretomes of CSCs and isogenic differentiated tumor cells (DTCs) isolated from three distinct metastasized colon tumors. Mass spectrometry-based proteomics identified 1254 unique proteins in the conditioned media of the paired CSC and DTC cultures. Ingenuity Pathway Analysis revealed that proteins governing 'Cell Death' were most significantly enriched in the CSC secretome. The vast majority of these (37/43) promote cell survival. The CSC secretome is also characterized by a pro-survival Nrf2 antioxidant signature. Interestingly, proteome-maintenance networks are highly enriched in the CSC secretome. CSCs also secrete high levels of drug-metabolizing enzymes, including aldehyde dehydrogenase 1 (ALDH1A1) and bleomycin hydrolase (BLMH). We show that these enzymes cause extracellular detoxification of maphosphamide and bleomycin respectively. We conclude that colorectal CSCs are characterized by extensive survival and anti-oxidant networks, which are likely to contribute to CSC-intrinsic drug-resistance. In addition, CSCs may modulate drug responses in nearby tumor cells by detoxifying chemotherapeutic drugs in the extracellular space., Biological Significance: Cancer stem cells are thought to play an important role in mediating drug resistance and tumor recurrence following chemotherapy. Therefore, it is important to identify the factors that are secreted by them. Our results provide novel insights into the pathways that govern the intrinsic resistance of CSCs to chemotherapy and, furthermore, demonstrate that they can also inactivate chemotherapeutic drugs in the extracellular space. A better understanding of the pathways that govern drug resistance in CSCs may help in developing effective CSC-targeting drugs., (© 2013.)

Background: Little is known about the factors that drive metastasis formation in colorectal cancer (CRC). Here, we set out to identify genes and proteins in patients with colorectal liver metastases that correlate with early disease recurrence. Such factors may predict a propensity for metastasis in earlier stages of CRC., Methods: Gene expression profiling and proteomics were used to identify differentially expressed genes/proteins in resected liver metastases that recurred within 6 months following liver surgery vs those that did not recur for >24 months. Expression of the identified genes/proteins in stage II (n=243) and III (n=176) tumours was analysed by immunohistochemistry on tissue microarrays. Correlation of protein levels with stage-specific outcome was assessed by uni- and multivariable analyses., Results: Both gene expression profiling and proteomics identified Maspin to be differentially expressed in colorectal liver metastases with early (<6 months) and prolonged (>24 months) time to recurrence. Immunohistochemical analysis of Maspin expression on tumour sections revealed that it was an independent predictor of time to recurrence (log-rank P=0.004) and CRC-specific survival (P=0.000) in stage III CRC. High Maspin expression was also correlated with mucinous differentiation. In stage II CRC patients, high Maspin expression did not correlate with survival but was correlated with a right-sided tumour location., Conclusion: High Maspin expression correlates with poor outcome in CRC after spread to the local lymph nodes. Therefore, Maspin may have a stage-specific function possibly related to tumour cell dissemination and/or metastatic outgrowth.

A key challenge in the data analysis of biological high-throughput experiments is to handle the often low number of samples in the experiments compared to the number of biomolecules that are simultaneously measured. Combining experimental data using independent technologies to illuminate the same biological trends, as well as complementing each other in a larger perspective, is one natural way to overcome this challenge. In this work we investigated if integrating proteomics and transcriptomics data from a brain cancer animal model using gene set based analysis methodology, could enhance the biological interpretation of the data relative to more traditional analysis of the two datasets individually. The brain cancer model used is based on serial passaging of transplanted human brain tumor material (glioblastoma--GBM) through several generations in rats. These serial transplantations lead over time to genotypic and phenotypic changes in the tumors and represent a medically relevant model with a rare access to samples and where consequent analyses of individual datasets have revealed relatively few significant findings on their own. We found that the integrated analysis both performed better in terms of significance measure of its findings compared to individual analyses, as well as providing independent verification of the individual results. Thus a better context for overall biological interpretation of the data can be achieved.

In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. Through the label-free LC-MS/MS analysis of fractionated samples, 1176 proteins were identified from culture filtrates of log phase and nutrient-starved cultures, and the protein levels of 230 proteins were increased in nutrient-starved culture filtrates, whereas those of 208 proteins were decreased. By means of Gene Ontology clustering analysis, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin-antitoxin systems were present in larger quantities in nutrient-starved cultures, supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy. Decreased abundance of proteins involved in amino acid and protein synthesis was apparent, as well as changes in the lipid metabolism. Further analysis of the dataset identified increased abundance of lipoproteins and decreased abundance of ESAT-6 family proteins. Results from the two-dimensional difference gel electrophoresis proteomics demonstrated overall agreement with the LC-MS/MS data and added complementary insights about protein degradation and modification.

In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin.

Colorectal cancer (CRC) is a major cause of cancer-related death in the western world. Screening to detect the disease in an early stage is the most effective approach to tackle this problem. In addition, better diagnostic tools for assessment of prognosis and prediction of response to drug therapy will allow for personalized therapies and better outcomes. Protein biomarkers that reflect tumor biology have the potential to address a wide range of clinical needs. These include diagnostic (screening) biomarkers for early detection, prognostic biomarkers for estimation of disease outcome, predictive biomarkers for adjuvant treatment stratification, and surveillance biomarkers for disease monitoring and treatment response. An important source for the discovery of potential biomarkers comes from mass spectrometry based proteomics research of the biology of CRC development. Here, we review recent colon cancer proteomics studies directed at identification of biomarker proteins. These include studies that use preclinical models (i.e. cell lines or murine tissues) as well as clinical materials (e.g. tissue and stool samples). We separately highlight some studies that focused on identification of cancer stem cell (CSC) related proteins in tumor spheroids, an in vitro model system for investigating CRC treatment response. Recent proteomics studies have generated many new candidate protein biomarkers. However, the lack of follow-up studies that lead to biomarker verification and/or validation remains a limiting factor in the translation of these candidate biomarkers into clinical applications. This is partly due to technological limitations which are bound to diminish with new technologies, including selected reaction monitoring mass spectrometry (SRM-MS). Antibodies are still required, though, both to perform high-throughput validation as well as to develop cost-effective tests for routine use in a clinical setting., (Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Hispanics in the United States represent diverse racial, ethnic, and socioeconomic groups, and manifest heterogeneous cardiovascular risks including diabetes. It is not known if there are residual differences in the control of diabetes among Hispanic groups given uniform access to diabetes care., Objective: To evaluate glucose control differences among Mexicans, Puerto Ricans, and Dominicans receiving substantial diabetes care and support in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial., Design: Secondary analysis of data from a randomized trial comparing two treatment strategies: intensive, targeting glycated hemoglobin below 6.0 %, and standard, targeting glycated hemoglobin between 7.0 % and 7.9 %., Participants: Seven hundred and sixteen Hispanic and 6066 non-Hispanic white participants were recruited from 77 clinical sites across the United States and Canada. There were 243 Mexicans, 199 Puerto Ricans, and 150 Dominicans; and 135 of these Hispanic groups were born in the United States., Main Measure: Glycated hemoglobin, Results: Compared to Puerto Ricans, Mexicans were more likely (HR=1.38, CI:0.90-2.10) and Dominicans as likely (HR=1.01, CI:0.66-1.54) to achieve glycated hemoglobin goal in the intensive arm. Participants born in the United States achieved glycated hemoglobin goal at a higher rate than those born elsewhere (HR=1.57, CI:0.99-2.51 in the intensive arm, HR=1.51, CI:0.95-2.43 in the standard arm). These differences were not statistically significant. In the intensive arm, Puerto Ricans (OR=0.47, CI:0.31-0.71), and Dominicans (OR=0.41, CI:0.26-0.66) were less likely than non-Hispanic whites to achieve glycated hemoglobin goal, whereas the difference between non-Hispanic whites and Mexicans was not statistically significant, (OR=0.66, CI:0.43-1.02)., Conclusions: Hispanic groups, given access to comprehensive diabetes care, differed from each other non-significantly and had a variable divergence from non-Hispanic whites in achieving intensive glycated hemoglobin goal. These differences, if confirmed, could be due to such factors as variable acculturation and functional health literacy levels that were not measured in the ACCORD trial, but should be further explored in future studies.

Motivation: Recent technology platforms in proteomics and genomics produce count data for quantitative analysis. Previous works on statistical significance analysis for count data have mainly focused on the independent sample setting, which does not cover the case where pairs of measurements are taken from individual patients before and after treatment. This experimental setting requires paired sample testing such as the paired t-test often used for continuous measurements. A state-of-the-art method uses a negative binomial distribution in a generalized linear model framework for paired sample testing. A paired sample design assumes that the relative change within each pair is constant across biological samples. This model can be used as an approximation to the true model in cases of heterogeneity of response in complex biological systems. We aim to specify the variation in response explicitly in combination with the inherent technical variation., Results: We formulate the problem of paired sample test for count data in a framework of statistical combination of multiple contingency tables. In particular, we specify explicitly a random distribution for the effect with an inverted beta model. The technical variation can be modeled by either a standard Poisson distribution or an exponentiated Poisson distribution, depending on the reproducibility of the acquisition workflow. The new statistical test is evaluated on both proteomics and genomics datasets, showing a comparable performance to the state-of-the-art method in general, and in several cases where the two methods differ, the proposed test returns more reasonable p-values., Availability: Available for download at http://www.oncoproteomics.nl/., Contact: t.pham@vumc.nl.

Macrophages are versatile cells, which phenotype is profoundly influenced by their environment. Pro-inflammatory classically activated or M1 macrophages, and anti-inflammatory alternatively-activated or M2 macrophages represent two extremes of a continuum of functional states. Consequently, macrophages that are present in tumors can exert tumor-promoting and tumor-suppressing activity, depending on the tumor milieu. In this study we investigated how human monocytes-the precursors of macrophages-are influenced by carcinoma cells of different origin. We demonstrate that monocytes, stimulated with breast cancer supernatant, showed increased expression of interleukin (IL)-10, IL-8 and chemokines CCL17 and CCL22, which are associated with an alternatively-activated phenotype. By contrast, monocytes that were cultured in supernatants of colon cancer cells produced more pro-inflammatory cytokines (e.g., IL-12 and TNFα) and reactive oxygen species. Secretome analysis revealed differential secretion of proteins by colon and breast cancer cell lines, of which the proteoglycan versican was exclusively secreted by colon carcinoma cell lines. Reducing active versican by blocking with monoclonal antibodies or shRNA diminished pro-inflammatory cytokine production by monocytes. Thus, colon carcinoma cells polarize monocytes toward a more classically-activated anti-tumorigenic phenotype, whereas breast carcinomas predispose monocytes toward an alternatively activated phenotype. Interestingly, presence of macrophages in breast or colon carcinomas correlates with poor or good prognosis in patients, respectively. The observed discrepancy in macrophage activation by either colon or breast carcinoma cells may therefore explain the dichotomy between patient prognosis and macrophage presence in these different tumors. Designing new therapies, directing development of monocytes toward M1 activated tumor macrophages in cancer patients, may have great clinical benefits.

The locus ceruleus is among the earliest affected brain regions in Parkinson's disease (PD) showing Lewy body pathology and neuronal loss. To improve our understanding of the pathogenesis of PD, we performed the first proteomic analysis ever of post-mortem locus ceruleus tissue of six pathologically confirmed PD patients, and six age- and gender-matched non-neurological controls. In total 2495 proteins were identified, of which 87 proteins were differentially expressed in the locus ceruleus of PD patients compared with controls. The majority of these differentially expressed proteins are known to be involved in processes that have been implicated in the pathogenesis of PD previously, including mitochondrial dysfunction, oxidative stress, protein misfolding, cytoskeleton dysregulation and inflammation. Several individual proteins were identified that have hitherto not been associated with PD, such as regucalcin, which plays a role in maintaining intracellular calcium homeostasis, and isoform 1 of kinectin, which is involved in transport of cellular components along microtubules. In addition, pathway analysis suggests a pathogenetic role for aminoacyl-tRNA-biosynthesis. These findings indicate that the proteome of the locus ceruleus of PD patients and non-neurological controls provides data that are relevant to the pathogenesis of PD, reflecting both known and potentially novel pathogenetic pathways., (© 2011 The Authors; Brain Pathology © 2011 International Society of Neuropathology.)

Breast cancer 1, early onset (BRCA1) hereditary breast cancer, a type of cancer with defects in the homology-directed DNA repair pathway, would benefit from the identification of proteins for diagnosis, which might also be of potential use as screening, prognostic, or predictive markers. Sporadic breast cancers with defects in the BRCA1 pathway might also be diagnosed. We employed proteomics based on one-dimensional gel electrophoresis in combination with nano-LC-MS/MS and spectral counting to compare the protein profiles of mammary tumor tissues of genetic mouse models either deficient or proficient in BRCA1. We identified a total of 3,545 proteins, of which 801 were significantly differentially regulated between the BRCA1-deficient and -proficient breast tumors. Pathway and protein complex analysis identified DNA repair and related functions as the major processes associated with the up-regulated proteins in the BRCA1-deficient tumors. In addition, by selecting highly connected nodes, we identified a BRCA1 deficiency signature of 45 proteins that enriches for homology-directed DNA repair deficiency in human gene expression breast cancer data sets. This signature also exhibits prognostic power across multiple data sets, with optimal performance in a data set enriched in tumors deficient in homology-directed DNA repair. In conclusion, by comparing mouse proteomes from BRCA1-proficient and -deficient mammary tumors, we were able to identify several markers associated with BRCA1 deficiency and a prognostic signature for human breast cancer deficient in homology-directed DNA repair.

Normal multipotent tissue stem cells (SCs) are the driving force behind tissue turnover and repair. The cancer stem cell theory holds that tumors also contain stem-like cells that drive tumor growth and metastasis formation. However, very little is known about the regulation of SC maintenance pathways in cancer and how these are affected by cancer-specific genetic alterations and by treatment. Proteomics is emerging as a powerful tool to identify the signaling complexes and pathways that control multi- and pluri-potency and SC differentiation. Here, the authors review the novel insights that these studies have provided and present a comprehensive strategy for the use of proteomics in studying cancer SC biology.

Background and Objective: Early detection of colon adenomas at high risk of progression and early-stage colorectal cancer (CRC) is an effective approach to reduce CRC death rates. Current screening methods lack specificity as they detect many adenomas that will never progress to CRC. The authors aimed to identify cell surface protein biomarkers with extracellular domains that could be targeted for molecular imaging and discriminate low-risk adenomas and normal colon from high-risk adenomas and CRC., Design: Cell surface proteins of five CRC cell lines were biotinylated, isolated and analysed by in-depth proteomics using gel electrophoresis and nanoliquid chromatography coupled to tandem mass spectrometry. Differential expression in adenomas and CRCs was based on mRNA expression and verified by immunohistochemical staining of tissue microarrays., Results: In total, 2609 proteins were identified in the cell surface fractions. Of these, 44 proteins were selected as promising cell surface candidate biomarkers for adenoma-to-carcinoma progression based on the following criteria: protein identification in at least four out of five cell lines, a predicted (trans)membrane location and increased mRNA expression in CRCs compared to adenomas. Increased protein expression in high-risk adenomas and CRCs compared to low-risk adenomas was confirmed by immunohistochemistry for glucose transporter type 1 (gene symbol SLC2A1; p<0.00001) and prion protein (gene symbol PRNP; p<0.005)., Conclusion: This study revealed glucose transporter type 1, prion protein and 42 other cell surface candidate biomarkers for adenoma-to-carcinoma progression that could potentially serve as targets for emerging molecular imaging modalities like optical imaging, ¹⁹F-MRI and positron emission tomography.

Purpose: Early detection of colorectal cancer (CRC) and its precursor lesions is an effective approach to reduce CRC mortality rates. This study aimed to identify novel protein biomarkers for the early diagnosis of CRC., Experimental Design: Proximal fluids are a rich source of candidate biomarkers as they contain high concentrations of tissue-derived proteins. The FabplCre;Apc(15lox/+) mouse model represents early-stage development of human sporadic CRC. Proximal fluids were collected from normal colon and colon tumors and subjected to in-depth proteome profiling by tandem mass spectrometry. Carcinoembryonic antigen (CEA) and CHI3L1 human serum protein levels were determined by ELISA., Results: Of the 2,172 proteins identified, quantitative comparison revealed 192 proteins that were significantly (P < 0.05) and abundantly (>5-fold) more excreted by tumors than by controls. Further selection for biomarkers with highest specificity and sensitivity yielded 52 candidates, including S100A9, MCM4, and four other proteins that have been proposed as candidate biomarkers for human CRC screening or surveillance, supporting the validity of our approach. For CHI3L1, we verified that protein levels were significantly increased in sera from patients with adenomas and advanced adenomas compared with control individuals, in contrast to the CRC biomarker CEA., Conclusion: These data show that proximal fluid proteome profiling with a mouse tumor model is a powerful approach to identify candidate biomarkers for early diagnosis of human cancer, exemplified by increased CHI3L1 protein levels in sera from patients with CRC precursor lesions., (©2012 AACR.)

For most diseases, better biomarkers are urgently needed to enable (early) detection, diagnosis, prognosis, stratification for therapy and response monitoring. Proteomics delineates gene products that carry out the majority of cellular functions, and thereby may not only yield insight into altered signaling pathways in disease, but also yield novel biomarkers. In recent years, great progress has been made in mass spectrometry-based analysis of clinical tissues and biofluids, with identification and quantification of thousands of proteins now becoming increasingly routine. However, biomarker validation and clinical translation has turned out to be challenging. In this review, we summarize current mass spectrometry-based proteomics strategies for biomarker discovery and verification using selected reaction monitoring, with a focus on progress and recent applications in clinical material using label-free approaches.

Purpose: Better breast cancer prognostication may improve selection of patients for adjuvant therapy. We conducted a retrospective longitudinal study in which we investigated sera of high-risk primary breast cancer patients, to search for proteins predictive of recurrence-free survival., Methods: Sera of 82 breast cancer patients obtained after surgery, but prior to the administration of adjuvant therapy, were fractionated using anion-exchange chromatography, to facilitate the detection of the low-abundant serum peptides. Selected fractions were subsequently analysed by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS), and the resulting protein profiles were searched for prognostic markers by appropriate bioinformatics tools., Results: Four peak clusters (i.e. m/z 3073, m/z 3274, m/z 4405 and m/z 7973) were found to bear significant prognostic value (P ≤ 0.01). The m/z 3274 candidate marker was structurally identified as inter-alpha-trypsin inhibitor heavy chain 4 fragment(658-688) in serum. Except for the m/z 7973 peak cluster, these peaks remained independently associated with recurrence-free survival upon multivariate Cox regression analysis, including clinical parameters of known prognostic value in this study population., Conclusion: Investigation of the postoperative serum proteome by, e.g., anion-exchange fractionation followed by SELDI-TOF MS analysis is promising for the detection of novel prognostic factors. However, regarding the rather limited study population, validation of these results by analysis of independent study populations is warranted to assess the true clinical applicability of discovered prognostic markers. In addition, structural identification of the other markers will aid in elucidation of their role in breast cancer prognosis, as well as enable development of absolute quantitative assays.

One of the proposed causes of obesity and metabolic syndrome is the excessive intake of products containing added sugars, in particular, fructose. Although the ability of excessive intake of fructose to induce metabolic syndrome is mounting, to date, no study has addressed whether a diet specifically lowering fructose but not total carbohydrates can reduce features of metabolic syndrome. A total of 131 patients were randomized to compare the short-term effects of 2 energy-restricted diets-a low-fructose diet vs a moderate natural fructose diet-on weight loss and metabolic syndrome parameters. Patients were randomized to receive 1500, 1800, or 2000 cal diets according to sex, age, and height. Because natural fructose might be differently absorbed compared with fructose from added sugars, we randomized obese subjects to either a low-fructose diet (<20 g/d) or a moderate-fructose diet with natural fruit supplements (50-70 g/d) and compared the effects of both diets on the primary outcome of weight loss in a 6-week follow-up period. Blood pressure, lipid profile, serum glucose, insulin resistance, uric acid, soluble intercellular adhesion molecule-1, and quality of life scores were included as secondary outcomes. One hundred two (78%) of the 131 participants were women, mean age was 38.8 ± 8.8 years, and the mean body mass index was 32.4 ± 4.5 kg/m(2). Each intervention diet was associated with significant weight loss compared with baseline. Weight loss was higher in the moderate natural fructose group (4.19 ± 0.30 kg) than the low-fructose group (2.83 ± 0.29 kg) (P = .0016). Compared with baseline, each intervention diet was associated with significant improvement in secondary outcomes. Reduction of energy and added fructose intake may represent an important therapeutic target to reduce the frequency of obesity and diabetes. For weight loss achievement, an energy-restricted moderate natural fructose diet was superior to a low-fructose diet., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: Treatment of local and distant head and neck cancer recurrences after radiotherapy remains an unsolved problem. In order to identify potential targets for use in effective therapy of recurrent tumors, we have investigated protein patterns in radioresistant (FaDu-IRR and SCC25-IRR, "IRR cells") as compared to parental (FaDu and SCC25) head and neck carcinoma cells., Methods and Materials: Radiation resistant IRR cells were derived from parental cells after repeated exposure to ionizing radiation 10 times every two weeks at a single dose of 10 Gy, resulting in a total dose of 100 Gy. Protein profiling in parental and IRR cells was carried out using two-dimensional differential gel electrophoresis (2D-DIGE) followed by MALDI-TOF/TOF mass spectrometry. Cell viability, cell migration assays and Western blot analysis were used to confirm results obtained using the proteome approach., Results: Forty-five proteins that were similarly modulated in FaDu-IRR and SCC25-IRR cells compared to parental cells were selected to analyze their common targets. It was found that these either up- or down-regulated proteins are closely related to the enhancement of cell migration which is regulated by Rac1 protein. Further investigations confirmed that Rac1 is up-regulated in IRR cells, and inhibiting its action reduces the migratory abilities of these cells. Additionally, the Rac1 inhibitor exerts cytostatic effects in HNSCC cells, mostly in migratory cells., Conclusions: Based on these results, we conclude that radioresistant HNSCC cells possess enhanced metastatic abilities that are regulated by a network of migration-related proteins. Rac1 protein may be considered as a putative biomarker of HNSCC radiation resistance, and as a potential therapeutic target for treating local and distant HNSCC recurrences., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Introduction: Body fluid biomarkers for clinical subtyping and monitoring of disease progression are of considerable interest in multiple sclerosis (MS). Proteomics tools are optimal for the unbiased simultaneous detection of large series of peptides and proteins., Objectives: To identify novel candidate biomarkers discriminating patients with MS from patients with other neurological diseases (OND), and for subtyping of relapsing-remitting (RR), secondary progressive (SP) and primary progressive (PP) MS patients using a high-throughput MALDI-TOF-based mass spectrometry method., Methods: Paired cerebrospinal fluid (CSF) and serum samples of 41 RRMS, 30 SPMS, 13 PPMS patients and 25 patients with OND were analysed., Results: Out of a total of 100 detected peptides in CSF and 200 peptides in serum, 11 peptides were differentially regulated in serum and two in CSF between patients with MS and the OND control group. Eleven peptides were differentially regulated in both serum and CSF between relapse-onset MS and PPMS patients. Lastly, four peptides were differentially regulated in serum and two in CSF between RRMS and SPMS patients. Specific peaks regulated in MS were tentatively identified as fragments of secretogranin III and complement C3. The peak intensity of the CSF peptide ion with m/z value 8607.7 correlated to atrophy (r = -0.27, p < 0.005), black hole volumes (r = 0.31, p < 0.008) and total lesion load (r = 0.34, p < 0.003). A serum peptide with m/z value of 872.4 elevated in SPMS correlated to Expanded Disability Status Scale (r = 0.341, p < 0.005) and atrophy (r = -0.286, p < 0.028)., Conclusions: Using high-throughput body fluid profiling by MALDI-TOF mass spectrometry, small proteins and peptides were detected as promising candidate biomarkers for diagnosis and disease progression of MS.

Background & Aims: Stem cells of normal tissues have resistance mechanisms that allow them to survive genotoxic insults. The stem cell-like cells of tumors are defined by their tumor-initiating capacity and may have retained these resistance mechanisms, making them resistant to chemotherapy. We studied the relationship between resistance to the topoisomerase I inhibitor irinotecan and tumor-initiating potential in human colonosphere cultures and in mice with colorectal xenograft tumors., Methods: Colonosphere cultures were established from human colorectal tumor specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. Stem cell and differentiation markers were analyzed by immunoblotting and fluorescence-activated cell sorting. Clone- and tumor-initiating capacities were assessed by single-cell cloning and in immune-deficient mice. Sensitivity to irinotecan was assessed in vitro and in tumor-bearing mice. The relationship between drug resistance and tumor-initiating capacity was tested by fluorescence-activated cell sorting of colonosphere cells, based on expression of ABCB1 and aldehyde dehydrogenase (ALDH) activity., Results: Colonosphere cultures had a high capacity to initiate tumors in mice and were resistant to irinotecan. Inhibition of the drug-efflux pump ABCB1 by PSC-833 allowed irinotecan to eradicate tumor-initiating cells. However, ABCB1 was expressed only by a subpopulation of differentiated tumor cells that did not form clones or tumors. Conversely, tumor-initiating cells were ABCB1-negative and were identified by high ALDH activity. Tumorigenic ALDHhigh/ABCB1negative cells generated nontumorigenic ALDHlow/ABCB1positive daughter cells in vitro and in tumor xenografts. PSC-833 increased the antitumor efficacy of irinotecan in mice., Conclusions: The resistance of colorectal tumors to irinotecan requires the cooperative action of tumor-initiating ALDHhigh/ABCB1negative cells and their differentiated, drug-expelling, ALDHlow/ABCB1positive daughter cells., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Detecting and removing high-risk adenomas and early colorectal cancer (CRC) can reduce mortality of this disease. The noninvasive fecal occult blood test (FOBT; guaiac-based or immunochemical) is widely used in screening programs and although effective, it leaves room for improvement in terms of test accuracy. Molecular tests are expected to be more sensitive, specific and informative than current detection tests, and are promising future tools for CRC screening. This review provides an overview of the performances of DNA, RNA, and protein markers for CRC detection in stool and blood. Most emphasis currently is on DNA and protein markers. Among DNA markers there is trend to move away from mutation markers in favor of methylation markers. The recent boost in proteomics research leads to many new candidate protein markers. Usually in small series, some markers show better performance than the present FOBT. Evaluation in large well-controlled randomized trials is the next step needed to take molecular markers for CRC screening to the next level and warrant implementation in a screening setting.

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in cerebrospinal fluid (CSF) are being used in clinical practice. Anti-aquaporin-4 antibodies in serum are currently useful for the diagnosis of neuromyelitis optica (NMO), but we could expect novel CSF biomarkers that help define prognosis and response to treatment for this disease. One of the most critical factors in biomarker research is the inadequate powering of studies performed by single centers. Collaboration between investigators is needed to establish large biobanks of well-defined samples. A key issue in collaboration is to establish standardized protocols for biobanking to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by pre-analytical factors. Here, consensus guidelines for CSF collection and biobanking are presented, based on the guidelines that have been published by the BioMS-eu network for CSF biomarker research. We focussed on CSF collection procedures, pre-analytical factors and high quality clinical and paraclinical information. Importantly, the biobanking protocols are applicable for CSF biobanks for research targeting any neurological disease.

Mass spectrometry can be used to generate diagnostic peptide peak profiles "signatures" of serum samples. Peak profiles can be used to compare different sera and correlate samples (i.e., patient groups) with clinical data to assist in diagnosis, monitoring, and/or prediction. We describe the semiautomated capture of serum peptides/small proteins with magnetic beads that harbor C18 alkyl chains, the deposition of captured material on a target plate for MALDI-TOF mass spectrometry, as well as, general guidelines for data acquisition. We also include, in a separate note, a short manual version of the capture procedure. Overall, the serum sample processing protocol, reported here, is reproducible and robust. In conjunction with MALDI-TOF mass spectrometry, this protocol allows for the profiling of several hundreds of serum peptides.

Colorectal cancer (CRC) is a common cause of cancer-related mortality in the developed world. Improved methods for early detection and disease management are urgently needed. Many efforts in the past 5 years have been devoted to protein biomarker discovery for early detection of CRC. Here, we discuss identity-based studies employing tandem mass spectrometry that analyzed clinical material as well as model systems. Through meta-analysis we provide a list of CRC-associated tissue proteins discovered in multiple studies, with the greater majority being 2D gel-based discoveries coupled to MS/MS. So far only a limited number of CRC-associated proteins have been validated in serum for non-invasive testing for CRC. This list includes several intracellular and nuclear proteins that a priori would not have been considered candidate biomarkers based on their predicted subcellular localization. Finally, we highlight promising new directions that combine targeted analyses of subcellular proteomes, like the cell surface, secretome, exosome, and nuclear matrix, with nanoLC-MS/MS-based proteomics. We anticipate that in the near future, these novel mass spectrometry-based in-depth approaches will uncover many novel, specific CRC marker candidates in clinical tissues and that their targeted validation with multi-reaction monitoring MS will speed up development of non-invasive tests in feces and serum/plasma., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The inaccuracy of the early diagnosis of Parkinson's disease (PD) has been a major incentive for studies aimed at the identification of biomarkers. Brain-derived cerebrospinal fluid (CSF) proteins are potential biomarkers considering the major role that proteins play in PD pathogenesis. In this review, we discuss the current hypotheses about the pathogenesis of PD and identify the most promising candidate biomarkers among the CSF proteins studied so far. The list of potential markers includes proteins involved in various pathogenetic processes, such as oxidative stress and protein aggregation. This list will undoubtedly grow in the near future by application of CSF proteomics and subsequent validation of identified proteins. Probably a single biomarker will not suffice to reach high sensitivity and specificity, because PD is pathogenetically heterogeneous and shares etiological factors with other neurodegenerative diseases. Furthermore, identified candidate biomarkers will have to be thoroughly validated before they can be implemented as diagnostic aids.

Purpose: For biomarker discovery in cerebrospinal fluid (CSF), removal of major serum proteins is advantageous as more CSF proteins including brain-derived proteins can be identified. Our goal was to create a reproducible discovery workflow with acceptable throughput that can identify 500-1000 CSF proteins in small volumes of CSF., Experimental Design: In this study, we compared the performance of two multi-affinity depletion methods in spin filter format: MARS Human 14 and Seppro-IgY-14. To this end, we analyzed depleted and bound CSF fractions isolated from 0.5 mL aliquots of the same CSF sample (n=3 per depletion method) by label-free GeLC-MS/MS-based proteomics and normalized spectral counting., Results: The whole CSF dataset contained 884 proteins identified at high confidence. Depletion spin filter performance was assessed in terms of sensitivity and reproducibility of the CSF analysis. MARS and IgY-14 spin filters yielded comparable reproducibility of protein identification (71-74%) and quantification (CV 17-18%) but a significant difference in the total number of identified CSF proteins (767 and 703 proteins, respectively)., Conclusions and Clinical Relevance: The MARS filter compared to IgY-14 filter provides a CSF analysis with enhanced proteome coverage. We anticipate that this enhanced sensitivity will facilitate biomarker discovery in early stages of cancer or neurological disease., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Abnormalities in nuclear phenotype and chromosome structure are key features of cancer cells. Investigation of the protein determinants of nuclear subfractions in cancer may yield molecular insights into aberrant chromosome function and chromatin organization and in addition may yield biomarkers for early cancer detection. Here we evaluate a proteomics work flow for profiling protein constituents in subnuclear domains in colorectal cancer tissues and apply this work flow to a comparative analysis of the nuclear matrix fraction in colorectal adenoma and carcinoma tissue samples. First, we established the reproducibility of the entire work flow. In a reproducibility analysis of three nuclear matrix fractions independently isolated from the same colon tumor homogenate, 889 of 1,047 proteins (85%) were reproducibly identified at high confidence (minimally two peptides per protein at 99% confidence interval at the protein level) with an average coefficient of variance for the number of normalized spectral counts per protein of 30%. This indicates a good reproducibility of the entire work flow from biochemical isolation to nano-LC-MS/MS analysis. Second, using spectral counting combined with statistics, we identified proteins that are significantly enriched in the nuclear matrix fraction relative to two earlier fractions (the chromatin-binding and intermediate filament fractions) isolated from six colorectal tissue samples. The total data set contained 2,059 non-redundant proteins. Gene ontology mining and protein network analysis of nuclear matrix-enriched proteins revealed enrichment for proteins implicated in "RNA processing" and "mRNA metabolic process." Finally, an explorative comparison of the nuclear matrix proteome in colorectal adenoma and carcinoma tissues revealed many proteins previously implicated in oncogenesis as well as new candidates. A subset of these differentially expressed proteins also exhibited a corresponding change at the mRNA level. Together, the results show that subnuclear proteomics of tumor tissue is feasible and a promising avenue for exploring oncogenesis.

Motivation: Spectral count data generated from label-free tandem mass spectrometry-based proteomic experiments can be used to quantify protein's abundances reliably. Comparing spectral count data from different sample groups such as control and disease is an essential step in statistical analysis for the determination of altered protein level and biomarker discovery. The Fisher's exact test, the G-test, the t-test and the local-pooled-error technique (LPE) are commonly used for differential analysis of spectral count data. However, our initial experiments in two cancer studies show that the current methods are unable to declare at 95% confidence level a number of protein markers that have been judged to be differential on the basis of the biology of the disease and the spectral count numbers. A shortcoming of these tests is that they do not take into account within- and between-sample variations together. Hence, our aim is to improve upon existing techniques by incorporating both the within- and between-sample variations., Result: We propose to use the beta-binomial distribution to test the significance of differential protein abundances expressed in spectral counts in label-free mass spectrometry-based proteomics. The beta-binomial test naturally normalizes for total sample count. Experimental results show that the beta-binomial test performs favorably in comparison with other methods on several datasets in terms of both true detection rate and false positive rate. In addition, it can be applied for experiments with one or more replicates, and for multiple condition comparisons. Finally, we have implemented a software package for parameter estimation of two beta-binomial models and the associated statistical tests., Availability and Implementation: A software package implemented in R is freely available for download at http://www.oncoproteomics.nl/., Supplementary Information: Supplementary data are available at Bioinformatics online.

Meningiomas are among the most frequent tumors of the brain and spinal cord accounting for 15-20% of all central nervous system tumors and frequently associated with neurofibromatosis type 2. In this study, we aimed to unravel molecular meningioma tumorigenesis and discover novel protein biomarkers for diagnostic and/or prognostic purposes and performed in-depth proteomic profiling of meningioma cells compared to human primary arachnoidal cells. We isolated proteins from meningioma cell line SF4433 and human primary arachnoidal cells and analyzed the protein profiles by Gel-nanoLC-MS/MS in conjunction with protein identification and quantification by shotgun nanoLC tandem mass spectrometry and spectral counting. Differential analysis of meningiomas revealed changes in the expression levels of 281 proteins (P < 0.01) associated with various biological functions such as DNA replication, recombination, cell cycle, and apoptosis. Among several interesting proteins, we focused on a subset of the highly significantly up-regulated proteins, the minichromosome maintenance (MCM) family. We performed subsequent validation studies by qRT-PCR in human meningioma tissue samples (WHO grade I, 14 samples; WHO grade II, 7 samples; and WHO grade III, 7 samples) compared to arachnoidal tissue controls (from fresh autopsies; 3 samples) and found that MCMs are highly and significantly up-regulated in human meningioma tumor samples compared to arachnoidal tissue controls. We found a significant increase in MCM2 (8 fold), MCM3 (5 fold), MCM4 (4 fold), MCM5 (4 fold), MCM6 (3 fold), and MCM7 (5 fold) expressions in meningiomas. This study suggests that MCM family proteins are up-regulated in meningiomas and can be used as diagnostic markers.

Malignant gliomas (glioblastoma multiforme) have a poor prognosis with an average patient survival under current treatment regimens ranging between 12 and 14 months. The tumors are characterized by rapid cell growth, extensive neovascularization, and diffuse cellular infiltration of normal brain structures. We have developed a human glioblastoma xenograft model in nude rats that is characterized by a highly infiltrative non-angiogenic phenotype. Upon serial transplantation this phenotype will develop into a highly angiogenic tumor. Thus, we have developed an animal model where we are able to establish two characteristic tumor phenotypes that define human glioblastoma (i.e. diffuse infiltration and high neovascularization). Here we aimed at identifying potential biomarkers expressed by the non-angiogenic and the angiogenic phenotypes and elucidating the molecular pathways involved in the switch from invasive to angiogenic growth. Focusing on membrane-associated proteins, we profiled protein expression during the progression from an invasive to an angiogenic phenotype by analyzing serially transplanted glioma xenografts in rats. Applying isobaric peptide tagging chemistry (iTRAQ) combined with two-dimensional LC and MALDI-TOF/TOF mass spectrometry, we were able to identify several thousand proteins in membrane-enriched fractions of which 1460 were extracted as quantifiable proteins (isoform- and species-specific and present in more than one sample). Known and novel candidate proteins were identified that characterize the switch from a non-angiogenic to a highly angiogenic phenotype. The robustness of the data was corroborated by extensive bioinformatics analysis and by validation of selected proteins on tissue microarrays from xenograft and clinical gliomas. The data point to enhanced intercellular cross-talk and metabolic activity adopted by tumor cells in the angiogenic compared with the non-angiogenic phenotype. In conclusion, we describe molecular profiles that reflect the change from an invasive to an angiogenic brain tumor phenotype. The identified proteins could be further exploited as biomarkers or therapeutic targets for malignant gliomas.

Deficiency of cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1(P3H1) has been reported in autosomal-recessive lethal or severe osteogenesis imperfecta (OI). CRTAP, P3H1, and cyclophilin B (CyPB) form an intracellular collagen-modifying complex that 3-hydroxylates proline at position 986 (P986) in the alpha1 chains of collagen type I. This 3-prolyl hydroxylation is decreased in patients with CRTAP and P3H1 deficiency. It was suspected that mutations in the PPIB gene encoding CyPB would also cause OI with decreased collagen 3-prolyl hydroxylation. To our knowledge we present the first two families with recessive OI caused by PPIB gene mutations. The clinical phenotype is compatible with OI Sillence type II-B/III as seen with COL1A1/2, CRTAP, and LEPRE1 mutations. The percentage of 3-hydroxylated P986 residues in patients with PPIB mutations is decreased in comparison to normal, but it is higher than in patients with CRTAP and LEPRE1 mutations. This result and the fact that CyPB is demonstrable independent of CRTAP and P3H1, along with reported decreased 3-prolyl hydroxylation due to deficiency of CRTAP lacking the catalytic hydroxylation domain and the known function of CyPB as a cis-trans isomerase, suggest that recessive OI is caused by a dysfunctional P3H1/CRTAP/CyPB complex rather than by the lack of 3-prolyl hydroxylation of a single proline residue in the alpha1 chains of collagen type I.

Background: Only a minority of patients with advanced non-small cell lung cancer (NSCLC) benefit from chemotherapy. Serum peptide profiling of NSCLC patients was performed to investigate patterns associated with treatment outcome.Using magnetic bead-assisted serum peptide capture coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), serum peptide mass profiles of 27 NSCLC patients treated with cisplatin-gemcitabine chemotherapy and bortezomib were obtained. Support vector machine-based algorithms to predict clinical outcome were established based on differential pre-treatment peptide profiles and dynamic changes in peptide abundance during treatment., Results: A 6-peptide ion signature distinguished with 82% accuracy, sensitivity and specificity patients with a relatively short vs. long progression-free survival (PFS) upon treatment. Prediction of long PFS was associated with longer overall survival. Inclusion of 7 peptide ions showing differential changes in abundance during treatment led to a 13-peptide ion signature with 86% accuracy at 100% sensitivity and 73% specificity. A 5-peptide ion signature could separate patients with a partial response vs. non-responders with 89% accuracy at 100% sensitivity and 83% specificity. Differential peptide profiles were also found when comparing the NSCLC serum profiles to those from cancer-free control subjects., Conclusion: This study shows that serum peptidome profiling using MALDI-TOF-MS coupled to pattern diagnostics may aid in prediction of treatment outcome of advanced NSCLC patients treated with chemotherapy.

Several parasitic helminthes, such as the human parasite Schistosoma mansoni, express glycoconjugates that contain terminal GalNAc beta1-4(Fuc alpha1-3)GlcNAc beta-R (LDNF) moieties. These LDNF glycans are dominant antigens of the parasite and are recognized by human dendritic cells via the C-type lectin DC-SIGN. To study the functional role of the LDNF antigen in interaction with the immune system, we have developed an easy chemoenzymatic method to synthesize multivalent neoglycoconjugates carrying defined amounts of LDNF antigens. An acceptor substrate providing a terminal N-acetylglucosamine was prepared by coupling a fluorescent hydrophobic aglycon, 2,6-diaminopyridine (DAP), to N,N'-diacetylchitobiose. By the subsequent action of recombinant Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase and human alpha1,3-fucosyltransferase VI (FucT-VI), this substrate was converted to the LDNF antigen. We showed that human FucT-VI has a relatively high affinity for the unusual substrate GalNAc beta1-4GlcNAc (LDN), and this enzyme was used to produce micromolar amounts of LDNF-DAP. The synthesized LDNF-DAP was coupled to carrier protein via activation of the DAP moiety by diethyl squarate. By varying the molar glycan:protein ratio, neoglycoconjugates were constructed with defined amounts of LDNF, as was determined by MALDI-TOF analysis and ELISA using an anti-LDNF antibody.

Aim: Novel diagnostic breast cancer markers have been extensively searched for in the proteome, using, among others, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Thus far, the majority of SELDI-TOF MS studies have investigated samples originating from biorepositories, which hampers biomarker discovery as they likely suffer from variable adherence to collection protocols., Material and Methods: We investigated breast cancer (n=75) and control (n=26) serum and tissue samples, collected prospectively by rigorous adherence to a strictly defined protocol. Sera were collected preoperatively and postoperatively, and serum and tissue samples were analyzed by SELDI-TOF MS using the IMAC30 Ni and Q10 pH 8 array., Results: Three serum peaks were significantly associated with breast cancer, while in tissue, 27 discriminative peaks were detected. Several peak clusters gradually increased or decreased in intensity from healthy to benign to cancer, or with increasing cancer stage. The constructed classification trees had a tenfold cross-validated performance of 67% to 87%. Two tissue peaks were identified as N-terminal albumin fragments. These are likely to have been generated by (breast) cancer-specific proteolytic activity in the tumor microenvironment., Conclusions: These albumin fragment scan potentially provide insights into the pathophysiological mechanisms associated with, or underlying, breast cancer, and aid in improving breast cancer diagnosis.

Apoptosis is a process of cellular suicide executed by caspases. Impaired activation of caspase-9 may contribute to chemoresistance in cancer. Activation of caspase-9 occurs after binding to Apaf-1 and formation of the apoptosome in the presence of cytochrome c/(d)ATP. We used a proteomics approach to identify proteins in caspase-9-protein complexes in extracts derived from NSCLC cells with(out) cytochrome c/dATP. Using co-immunoprecipitation, one-dimensional gel electrophoresis and tandem mass spectrometry, 38 proteins were identified of which 24 differential interactors. The differential interactors can be functionally assigned to cytoskeletal (re)organization and cell motility, catalytic activity, and transcriptional processes and apoptosis. The interaction of caspase-9 with Apaf-1 was confirmed and acetylserotonin-O-methyltransferase-like protein was identified as a candidate substrate of caspase-9. Novel interactors were found including galectin-3, swiprosin-1 and the membrane-cytoskeleton linkers Ezrin/Radixin/Moesin. Co-immunoprecipitation and Western blot experiments confirmed the interaction of caspase-9 with several identified binding partners. A large number of cytoskeletal proteins associated with unprocessed caspase-9 may indicate a scaffold function of this structure and/or may act as caspase substrates during apoptosis. Together, our results indicate that proteomic analysis of the caspase-9-associated protein complexes is a powerful exploratory approach to identify novel caspase substrates and/or regulators of caspase-9-dependent apoptosis.

The nuclear matrix (NM) model posits the presence of a protein/RNA scaffold that spans the mammalian nucleus. The NM proteins are involved in basic nuclear function and are a promising source of protein biomarkers for cancer. Importantly, the NM proteome is operationally defined as the proteins from cells and tissue that are extracted following a specific biochemical protocol; in brief, the soluble proteins and lipids, cytoskeleton, and chromatin elements are removed in a sequential fashion, leaving behind the proteins that compose the NM. So far, the NM has not been sufficiently verified as a biological entity and only preliminary at the molecular level. Here, we argue for a combined effort of proteomics, immunodetection and microscopy to unravel the composition and structure of the NM.

The revolution in genomics and proteomics has produced complex technologies enabling an insight into the functional effectors of cellular processes. In oncology these technologies lead to the identification of biological markers which may provide the starting point for the development and identification of diagnostic tests and therapeutic targets. To identify and validate reliable tumor markers within the proteome, it is necessary, prior to tandem mass spectrometry, to reduce sample complexity. This can be done by robust fractionation and separation techniques. This review addresses the discovery stage of onco-proteomics - the strategies for target identification and biomarker discovery in solid tumors and biofluids. The overview includes different proteomic methods, from gel-based to liquid chromatography (LC)-based separations of proteins/peptides, and the corresponding detection by mass spectrometry. The quantitative methods in mass spectrometry include techniques based on stable isotope labeling of proteins/peptides and label-free methods. A particular emphasis is given to proteomics-based biomarker discovery in biofluids (e.g. plasma, urine, secretome, cerebrospinal fluid) and target identification in tissue for anti-angiogenic therapies.