A well‐characterised peak identification list of MALDI MS profile peaks for human blood serum

MALDI MS profiling, using easily available body fluids such as blood serum, has attracted considerable interest for its potential in clinical applications. Despite the numerous reports on MALDI MS profiling of human serum, there is only scarce information on the identity of the species making up these profiles, particularly in the mass range of larger peptides. Here, we provide a list of more than 90 entries of MALDI MS profile peak identities up to 10 kDa obtained from human blood serum. Various modifications such as phosphorylation were detected among the peptide identifications. The overlap with the few other MALDI MS peak lists published so far was found to be limited and hence our list significantly extends the number of identified peaks commonly found in MALDI MS profiling of human blood serum.     

Physiological,biochemical, and proteome profiling reveals key pathways underlying the drought stress responses of Hippophae rhamnoides

The effects of drought on plant growth and development are occurring as a result of climate change and the growing scarcity of water resources. Hippophae rhamnoides has been exploited for soil and water conservation for many years. However, the outstanding drought‐resistance mechanisms possessed by this species remain unclear. The protein, physiological, and biochemical responses to medium and severe drought stresses in H. rhamnoides seedlings are analyzed. Linear decreases in photosynthesis rate, transpiration rate, and the content of indole acetic acid in roots, as well as a linear increase in the contents of abscisic acid, superoxide dismutase, glutathione reductase, and zeatin riboside in leaves are observed as water potential decreased. At the same time, cell membrane permeability, malondialdehyde, stomatal conductance, water use efficiency, and contents of zeatin riboside in roots and indole acetic acid in leaves showed nonconsistent changes. DIGE and MS/MS analysis identified 51 differently expressed protein spots in leaves with functions related to epigenetic modification and PTM in addition to normal metabolism, photosynthesis, signal transduction, antioxidative systems, and responses to stimuli. This study provides new insights into the responses and adaptations in this drought‐resistant species and may benefit future agricultural production.     

Comparative proteomic and transcriptomic profiling of the human hepatocellular carcinoma

Proteome analysis of human hepatocellular carcinoma (HCC) was done using two-dimensional difference gel electrophoresis. To gain an understanding of the molecular events accompanying HCC development, we compared the protein expression profiles of HCC and non-HCC tissue from 14 patients to the mRNA expression profiles of the same samples made from a cDNA microarray. A total of 125 proteins were identified, and the expression profiles of 93 proteins (149 spots) were compared to the mRNA expression profiles. The overall protein expression ratios correlated well with the mRNA ratios between HCC and non-HCC (Pearson’s correlation coefficient: r = 0.73). Particularly, the HCC/non-HCC expression ratios of proteins involved in metabolic processes showed significant correlation to those of mRNA (r = 0.9). A considerable number of proteins were expressed as multiple spots. Among them, several proteins showed spot-to-spot differences in expression level and their expression ratios between HCC and non-HCC poorly correlated to mRNA ratios. Such multi-spotted proteins might arise as a consequence of post-translational modifications.     

Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.     

Proteome analysis reveals adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung environment

Pseudomonas aeruginosa is known for the chronic lung colonization of cystic fibrosis (CF) patients in addition to eye, ear and urinary tract infections. With the underlying disease CF patients are predisposed to P. aeruginosa chronic lung infection, which leads to morbidity and mortality. In this study, we compared the protein expression profile of a CF lung-adapted P. aeruginosa strain C with that of the burn-wound isolate PAO. Differentially expressed proteins from the whole-cell, membrane, periplasmic as well as extracellular fraction were identified. The whole-cell proteome of strain C showed down-regulation of several proteins involved in amino acid metabolism, fatty acid metabolism, energy metabolism and adaptation leading to a highly distinct proteome pattern for strain C in comparison to PAO. Analysis of secreted proteins by strain C compared to PAO revealed differential expression of virulence factors under non-inducing conditions. The membrane proteome of strain C showed modulation of the expression of porins involved in nutrient and antibiotic influx. The proteome of the periplasmic space of strain C showed retention of elastase despite that the equal amounts were secreted by strain C and PAO. Altogether, our results elucidate adaptive strategies of P. aeruginosa towards the nutrient-rich CF lung habitat during the course of chronic colonization.     

Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers

We developed a practical strategy for serum protein profiling using antibody microarrays and applied the method to the identification of potential biomarkers in prostate cancer serum. Protein abundances from 33 prostate cancer and 20 control serum samples were compared to abundances from a common reference pool using a two-color fluorescence assay. Robotically spotted microarrays containing 184 unique antibodies were prepared on two different substrates: polyacrylamide based hydrogels on glass and poly-1-lysine coated glass with a photoreactive cross-linking layer. The hydrogel substrate yielded an average six-fold higher signal-to-noise ratio than the other substrate, and detection of protein binding was possible from a greater number of antibodies using the hydrogels. A statistical filter based on the correlation of data from "reverse-labeled" experiment sets accurately predicted the agreement between the microarray measurements and enzyme-linked immunosorbent assay measurements, showing that this parameter can serve to screen for antibodies that are functional on microarrays. Having defined a set of reliable microarray measurements, we identified five proteins (von Willebrand Factor, immunoglobulinM, Alpha1-antichymotrypsin, Villin and immunoglobulinG) that had significantly different levels between the prostate cancer samples and the controls. These developments enable the immediate use of high-density antibody and protein microarrays in biomarker discovery studies.     

LC-MS-based serum metabolic profiling for genitourinary cancer classification and cancer type-specific biomarker discovery

Bladder cancer (BC) and kidney cancer (KC) are the first two commonly occurring genitourinary cancers in China. In this study, a comprehensive LC-MS-based method, which utilizes both reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) separations, has been carried out in conjunction with multivariate data analysis to discriminate the global serum profiles of BC, KC, and noncancer controls. An independent test set consisting of different patients has been used to objectively evaluate the predictive ability of the analysis platform. Excellent sensitivity and specificity have been achieved in detection of KC and BC. The results suggest that serum metabolic profiling could be used for different types of genitourinary cancer diagnosis. Furthermore, cancer type-specific biomarkers were found through a critical selection criterion. As a result, eicosatrienol, azaprostanoic acid, docosatrienol, retinol, and 14'-apo-beta-carotenal were found as specific biomarkers for BC; and PE(P-16:0e/0:0), glycerophosphorylcholine, ganglioside GM3 (d18:1/22:1), C17 sphinganine, and SM(d18:0/16:1(9Z)) were found as specific biomarkers for KC. Receiver operating characteristic (ROC) analysis was used for the preliminary evaluation of the biomarkers. These biomarkers have great potential to be used in the clinical diagnosis after further rigorous assessment.     

Proteome profiling of Populus euphratica Oliv. upon heat stress

BACKGROUND AND AIMS: Populus euphratica is a light-demanding species ecologically characterized as a pioneer. It grows in shelter belts along riversides, being part of the natural desert forest ecosystems in China and Middle Eastern countries. It is able to survive extreme temperatures, drought and salt stress, marking itself out as an important plant species to study the mechanisms responsible for survival of woody plants under heat stress. METHODS: Heat effects were evaluated through electrolyte leakage on leaf discs, and LT(50) was determined to occur above 50 degrees C. Protein accumulation profiles of leaves from young plants submitted to 42/37 degrees C for 3 d in a phytotron were determined through 2D-PAGE, and a total of 45 % of up- and downregulated proteins were detected. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)/TOF analysis, combined with searches in different databases, enabled the identification of 82 % of the selected spots. KEY RESULTS: Short-term upregulated proteins are related to membrane destabilization and cytoskeleton restructuring, sulfur assimilation, thiamine and hydrophobic amino acid biosynthesis, and protein stability. Long-term upregulated proteins are involved in redox homeostasis and photosynthesis. Late downregulated proteins are involved mainly in carbon metabolism. CONCLUSIONS: Moderate heat response involves proteins related to lipid biogenesis, cytoskeleton structure, sulfate assimilation, thiamine and hydrophobic amino acid biosynthesis, and nuclear transport. Photostasis is achieved through carbon metabolism adjustment, a decrease of photosystem II (PSII) abundance and an increase of PSI contribution to photosynthetic linear electron flow. Thioredoxin h may have a special role in this process in P. euphratica upon moderate heat exposure.     

Comparative protein profiling of serum and plasma using an antibody suspension bead array approach

In the pursuit towards a systematic analysis of human diseases, array‐based approaches within antibody proteomics offer high‐throughput strategies to discover protein biomarkers in serum and plasma. To investigate the influence of sample preparation on such discovery attempts, we report on a systematic effort to compare serum and plasma protein profiles determined with an antibody suspension bead array. The intensity levels were used to define protein profiles and no significant differences between serum and plasma were observed for 79% of the 174 antibodies (targeting 156 proteins). By excluding 36 antibodies giving rise to differential intensity levels, cluster analysis revealed donor‐specific rather than preparation‐dependent grouping. With a cohort from a clinically relevant medical condition, the metabolic syndrome, the influence of the sample type on a multiplexed biomarker discovery approach was further investigated. Independent comparisons of protein profiles in serum and plasma revealed an antibody targeting ADAMTSL‐4, a protein that would qualify to be studied further in association with the condition. In general, the preparation type had an impact on the results of the applied antibody suspension bead array, and while the technical variability was equal, plasma offered a greater biological variability and allowed to give rise to more discoveries than serum.     

Robust substrate profiling method reveals striking differences in specificities of serum and lung fluid proteases

Proteases are candidate biomarkers and therapeutic targets for many diseases. Sensitive and robust techniques are needed to quantify proteolytic activities within the complex biological milieu. We hypothesized that a combinatorial protease substrate library could be used effectively to identify similarities and differences between serum and bronchoalveolar lavage fluid (BALF), two body fluids that are clinically important for developing targeted therapies and diagnostics. We used a concise library of fluorogenic probes to map the protease substrate specificities of serum and BALF from guinea pigs. Differences in the proteolytic fingerprints of the two fluids were striking: serum proteases cleaved substrates containing cationic residues and proline, whereas BALF proteases cleaved substrates containing aliphatic and aromatic residues. Notably, cleavage of proline-containing substrates dominated all other protease activities in both human and guinea pig serum. This substrate profiling approach provides a foundation for quantitative comparisons of protease specificities between complex biological samples.     

Plasticity of fibroblasts demonstrated by tissue-specific and function-related proteome profiling

Background

Fibroblasts are mesenchymal stromal cells which occur in all tissue types. While their main function is related to ECM production and physical support, they are also important players in wound healing, and have further been recognized to be able to modulate inflammatory processes and support tumor growth. Fibroblasts can display distinct phenotypes, depending on their tissue origin, as well as on their functional state.

Results

In order to contribute to the proteomic characterization of fibroblasts, we have isolated primary human fibroblasts from human skin, lung and bone marrow and generated proteome profiles of these cells by LC-MS/MS. Comparative proteome profiling revealed characteristic differences therein, which seemed to be related to the cell’s tissue origin. Furthermore, the cells were treated in vitro with the pro-inflammatory cytokine IL-1beta. While all fibroblasts induced the secretion of Interleukins IL-6 and IL-8 and the chemokine GRO-alpha, other inflammation-related proteins were up-regulated in an apparently tissue-dependent manner. Investigating fibroblasts from tumorous tissues of skin, lung and bone marrow with respect to such inflammation-related proteins revealed hardly any conformity but rather individual and tumor type-related variations. However, apparent up-regulation of IGF-II, PAI-1 and PLOD2 was observed in melanoma-, lung adenocarcinoma- and multiple myeloma-associated fibroblasts, as well as in hepatocellular carcinoma-associated fibroblasts.

Conclusions

Inflammation-related proteome alterations of primary human fibroblasts were determined by the analysis of IL-1beta treated cells. Tumor-associated fibroblasts from different tissue types hardly showed signs of acute inflammation but displayed characteristic functional aberrations potentially related to chronic inflammation. The present data suggest that the state of the tumor microenvironment is relevant for tumor progression and targeted treatment of tumor-associated fibroblasts may support anti-cancer strategies.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-41) contains supplementary material, which is available to authorized users.     

Microarray profiling of monocytic differentiation reveals miRNA-mRNA intrinsic correlation

MiRNAs (microRNAs) are small non-coding RNAs involved in mammalian gene expression of cellular processes including differentiation, apoptosis and cancer development. Both specific miRNAs and mRNAs have been identified during monocytic differentiation, but their interactions have not been fully characterized. Here we report that by genome-wide microarray analysis for U937 monocytic differentiation induced by TPA, a large number of miRNAs and mRNAs were differentially expressed, and by bioinformatics analysis could demonstrate that their functional pathway patterns overlap strongly. While expected negative correlation between the expression levels of miRNAs and their target mRNAs was seen, several positive correlations between miRNAs and host mRNAs were also observed, such as C13orf25/miR17, MCM7/miR93, and MGC14376/miR22. These microarray data were verified by quantitative RT-PCR, and the TPA-induced differentiation of U937 cells was confirmed by flow cytometric analysis. Our study suggests an intrinsic correlation between miRNAs and mRNAs underlying their interactions which would provide new insights for defining the mechanisms occurring during monocytic differentiation.     

Evaluation and optimization of IgY spin column technology in the depletion of abundant proteins from human serum

Serum depletion strategies are commonly implemented in order to remove abundant proteins, increasing the number of proteins detected in a biomarker study. The IgY spin columns used in this study bind 12 and 14 primate proteins, respectively. 1‐D SDS‐PAGE and 2‐DE revealed a suboptimal performance of the IgY spin columns. However, modification of the manufacturer's protocol, subjecting samples to two rounds of depletion, improved the number of proteins resolved by 2‐DE. With alteration of the manufacturer protocol, the Seppro^® IgY14 spin column can produce depleted serum with an increased number of spots resolved by 2‐DE compared to untreated serum.     

Prevalence of metabolic syndrome and gender differences

In a comparative study, involving 500 subjects with 294 males and 206 females aged 30 years and above, data were collected from NIMS (National Institute of Medical Sciences) hospital and research centre and controls from the general population whose age and sex were matched with subjects during the years 2010 - 2011. Metabolic syndrome was present both in women and men corresponding to 29% and 23% of the women's and men's sample, respectively. The prevalence was higher in women than in men. In women, elevated BMI, low HDL cholesterol, increased waist circumference and hyperglycemia were significantly larger contributors to the metabolic syndrome while in men these were hypertension and elevated triglycerides. The contribution of several metabolic components to the metabolic syndrome is different in men and women. This might contribute to gender specific differences in the relative risk of metabolic complications such as insulin resistance.     

Glycoform composition profiling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of flight-mass spectrometry

Pools of O-glycopeptides prepared from trypsin-digested reduced and alkylated human serum IgA1 have been analyzed using matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-ToF-MS) in the positive-ion mode, using 2,4,6-trihydroxy acetophenone-ammonium citrate matrix. Dozens of such pools prepared from normal serum IgA1 and from serum of patients with a number of different medical conditions have been routinely analyzed in this manner. The glycopeptides present in these pools possess identical amino acid sequences but are substituted with a variety of neutral and sialylated glycans and the spectra obtained were such that individual compositional glycoforms were baseline resolved. In addition, the spectra were reproducible, exhibiting a relative peak intensity and area variation of around 11-16%, enabling the technique to be used for the relative quantitation of the different compositional glycoforms present. This could be achieved manually or by applying a Java program especially developed for this purpose. The MS analysis described here is a major improvement over present MALDI methods used for profiling the O-glycosylation of IgA1. The MS methodology together with the Java data analysis are expected to be generally applicable for profiling O-linked glycopeptides derived from other glycoproteins and probably for N-linked glycopeptide pools.     

Comparative hepatic proteome analysis between lean and obese rats fed a high-fat diet reveals the existence of gender differences

Gender differences in obesity stem from metabolic and hormonal differences between sexes and contribute to differences between women and men in health risks attributable to obesity. We hypothesized that liver may be an ideal target for the evaluation of gender differences in obesity development in response to a high-fat diet (HFD). Therefore, to test this hypothesis, we performed a global proteome analysis in the liver of lean and obese rats of both genders who were fed an HFD through 2-DE combined with MALDI-TOF-MS. When rats were exposed to HFD, male rats gained more body weight with increased values of plasma biochemical parameters than female rats. Image analysis and further statistical analysis of a 2-DE protein map allowed for the detection and identification of 34 proteins that were significantly modulated in a gender-dependent manner. We found 19 proteins showing identical gender-different regulation in both normal diet (ND) and HFD. Five proteins also showed clear gender differences in both ND and HFD; however, their regulation modes in HFD were opposite to those in ND. Of particular interest, 10 proteins showed gender differences only in either ND or HFD rats. Present proteomic insight into gender-dimorphic protein modulation in liver would aid in the improvement of gender awareness in the health-care system and in implementation of evidence-based gender-specific clinical recommendations.     

蛋白质组学揭示美洲大蠊卵鞘在胚胎发育中的重要功能

作为世界性的卫生害虫,美洲大蠊Periplaneta americana对环境有惊人的适应性和极强的繁殖力,其强大的繁殖能力以及卵鞘对胚胎发育的有效保护,是其防治困难的内在原因.本研究以美洲大蠊卵鞘为研究对象,利用Label-free定量技术提取前期和中期的美洲大蠊卵鞘蛋白,检验合格后的蛋白溶液通过酶解,液质联用检测技...     

Gender differences in CMS and the effects of antidepressant venlafaxine in rats

Gender differences in susceptibility to chronic mild stress (CMS) and effects of venlafaxine in rats have been investigated in the current study. Male and female SD rats were exposed to CMS or CMS plus chronic venlafaxine administration (10 mg/kg, 21 days) in order to study depressive behavior in rats. Rats were tested in open field test and sucrose preference test to figure out gender differences in behavior. Then serum corticosterone and the expression of FKBP5 in hippocampus of rats were detected to explore the possible mechanism. The results showed that the CMS impact on behavioral parameters and corticosterone levels and response to venlafaxine were gender dependent. Female rats appeared more vulnerable in the dysregulation of HPA axis to CMS. Venlafaxine treatment normalized depressive-like behavior in both gender. However, venlafaxine treated male rats exhibited better improved explore behavior and anhedonia. FKBP5 might be involved in the explanation of gender differences in CMS and venlafaxine treatment. Male and female rats respond differently to chronic stress and venlafaxine continuous treatment. This results have guiding meaning in design of trials related to stress induced depression.     

Gene expression profiling reveals two separate mechanisms regulating apoptosis in rectal carcinomas in vivo

The level of apoptosis in rectal carcinomas of patients treated by surgery only predicts local failure; patients with intrinsically high-apoptotic tumors develop less local recurrences than patients with low levels of apoptosis. To identify genes involved in this intrinsic apoptotic process in vivo, 47 rectal tumors with known apoptotic phenotype (24 low- and 23 high-apoptotic) were analyzed by oligonucleotide microarray technology. We identified several genes differentially expressed between low- and high-apoptotic tumors. Unsupervised clustering of the tumors based on expression levels of these genes separated the low-apoptotic from the high-apoptotic tumors, indicating a gene expression-dependent regulation. In addition, this clustering revealed two subgroups of high-apoptotic tumors. One high-apoptotic subgroup showed subtle differences in mRNA and protein expression of the known apoptotic regulators BAX, cIAP2 and ARC compared to the low-apoptotic tumors. The other subgroup of high-apoptotic tumors showed high expression of immune-related genes; predominantly HLA class II and chemokines, but also HLA class I and interferon-inducible genes were highly expressed. Immunohistochemistry revealed HLA-DR expression in epithelial tumor cells in 70% of these high-apoptotic tumors. The expression data suggest that high levels of apoptosis in rectal carcinoma patients can be the result of either slightly altered expression of known pro- and anti-apoptotic genes or high expression of immune-related genes.