Proteomic analysis of anaplastic lymphoma cell lines: identification of potential tumour markers

Anaplastic large-cell lymphomas (ALCL) are high grade lymphomas of T or null phenotype often associated with the t(2;5) translocation leading to the expression of a chimeric protein consisting of the N-terminal portion of nucleophosmin (NPM) and the intracellular domain of the anaplastic lymphoma kinase (ALK). Although ALCL are recognized as distinct clinical, biological and cytogenetic entities, heterogeneities persist in this group of tumours, which exhibit a broad spectrum of morphological features. Particularly, the common type tumour consisting in large cells contrast with the small cell variant that is sometimes associated with a leukemic phase. The ALK-negative ALCL is often associated with a poor prognosis. Here, we investigated the proteome of these subtypes of tumours using patient-derived cell lines. We compared the proteome of the cytosolic fraction of NPM-ALK-positive versus NPM-ALK-negative cells on one hand, and the proteome of common cell type versus small cell variant on the other hand. The identification of a set of proteins differentially expressed in the subtypes of ALCL points to new diagnosis/prognosis markers. This study also provides interesting information on the molecular mechanisms responsible for the different subtypes of ALCL.     

Proteomic analysis reveals perturbed energy metabolism and elevated oxidative stress in hearts of rats with inborn low aerobic capacity

Selection on running capacity has created rat phenotypes of high-capacity runners (HCRs) that have enhanced cardiac function and low-capacity runners (LCRs) that exhibit risk factors of metabolic syndrome. We analysed hearts of HCRs and LCRs from generation 22 of selection using DIGE and identified proteins from MS database searches. The running capacity of HCRs was six-fold greater than LCRs. DIGE resolved 957 spots and proteins were unambiguously identified in 369 spots. Protein expression profiling detected 67 statistically significant (p<0.05; false discovery rate <10%, calculated using q-values) differences between HCRs and LCRs. Hearts of HCR rats exhibited robust increases in the abundance of each enzyme of the β-oxidation pathway. In contrast, LCR hearts were characterised by the modulation of enzymes associated with ketone body or amino acid metabolism. LCRs also exhibited enhanced expression of antioxidant enzymes such as catalase and greater phosphorylation of α B-crystallin at serine 59, which is a common point of convergence in cardiac stress signalling. Thus, proteomic analysis revealed selection on low running capacity is associated with perturbations in cardiac energy metabolism and provided the first evidence that the LCR cardiac proteome is exposed to greater oxidative stress.     

Proteomic analysis of the effects of the immunomodulatory mycotoxin deoxynivalenol

The mycotoxin deoxynivalenol (DON) contaminates cereals worldwide and is a common contaminant in the Western European diet. At high doses, DON induces acute gastrointestinal toxicity; chronic, low‐dose effects in humans are not well described, but immunotoxicity has been reported. In this study, 2‐DE was used to identify proteomic changes in human B (RPMI1788) and T (JurkatE6.1) lymphocyte cell lines after exposure to minimally toxic concentrations (up to 500 ng/mL) for 24 h. Proteins which changed their abundance post treatment, by a greater than 1.4‐fold change reproducible in three separate experiments consisting of 36 gels in total, are ubiquitin carboxyl‐terminal hydrolase isozyme L3, proteasome subunit β type‐4 and α type‐6, inosine‐5′‐monophosphate dehydrogenase 2, GMP synthase, microtubule‐associated protein RP/EB family member 1 (EB1), RNA polymerases I, II, III subunit ABC1, triosephosphate isomerase and transketolase. Flow cytometry was used to validate changes to protein expression, except for EB1. These findings provide insights as to how low‐dose exposure to DON may affect human immune function and may provide mechanism‐based biomarkers for DON exposure.     

Isatin‐binding proteins of rat and mouse brain: Proteomic identification and optical biosensor validation

Isatin (indole‐2,3‐dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Isatin itself and its analogues exhibit a wide range of pharmacological activities but its specific biological targets still are not well characterized. Affinity chromatography of Triton X‐100 lysates of soluble and particulate fractions of mouse and rat whole brain homogenates on 5‐aminocaproyl‐isatin‐Sepharose followed by subsequent proteomic analysis resulted in identification of 65 and 64 individual proteins, respectively. Isatin‐binding capacity of some of the identified proteins has been validated in an optical biosensor study using a Biacore 3000 optical biosensor, 5‐aminocarproyl‐isatin, and 5‐aminoisatin as the affinity ligands. The K_d values (of 0.1–20 μM) obtained during the optical biosensor experiments were consistent with the range of K_d values recently reported for [³H]isatin binding to brain sections. Although the number of isatin‐binding proteins identified in the mouse and rat brain was similar, only 21 proteins (about one‐third) were identical in the two species. This may be one reason for the differences in isatin effects in rats and mice reported in the literature.     

Proteomic and bioinformatic analysis of membrane proteome in type 2 diabetic mouse liver

Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study, we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT‐ICR MS, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, six proteins are associated with energy stress‐induced, nine proteins with inflammatory stress‐induced, and 14 proteins with endoplasmic reticulum stress‐induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a c‐Jun amino‐terminal kinase‐dependent manner. In addition, three proteins, 14–3‐3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD‐95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomics approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases.     

Proteomic analysis of fetal programming‐related obesity markers

The objectives of this study were to analyze fetal programming in rat brain using proteomic analysis and to identify fetal programming‐related obesity markers. Sprague–Dawley rats were divided into four feeding groups: (i) the Ad Libitum (AdLib)/AdLib group was given a normal diet during pregnancy and the lactation period; (ii) the AdLib/maternal food restriction group (FR) was subjected to 50% FR during the lactation period; (iii) the FR/AdLib group was subjected to 50% FR during pregnancy; and (iv) the FR/FR group was subjected to 50% FR during pregnancy and the lactation period. Offspring from each group were sacrificed at 3 weeks of age and whole brains were dissected. To obtain a maximum number of protein markers related to obesity, 2DE and Pathway Studio bioinformatics analysis were performed. The identities of the markers among the selected and candidate proteins were confirmed by Western blotting and immunohistochemistry. Proteomic and bioinformatics analyses revealed that expression of ubiquitin carboxy‐terminal hydrolase L1 (UCHL1) and Secernin 1 (SCRN1) were significantly different in the FR/AdLib group compared with the AdLib/AdLib group for both male and female offspring. These findings suggest that UCHL1 and SCRN1 may be used as fetal programming‐related obesity markers.     

Proteome map of the neural complex of the tunicate Ciona intestinalis,the closest living relative to vertebrates

Ciona intestinalis (the common sea squirt) is the closest living chordate relative to vertebrates with cosmopolitan presence worldwide. It has a relatively simple nervous system and development, making it a widely studied alternative model system in neuroscience and developmental biology. The use of Ciona as a model organism has increased significantly after the draft genome was published. In this study, we describe the first proteome map of the neural complex of C. intestinalis. A total of 544 proteins were identified based on 1DE and 2DE FTMS/ITMSMS analyses. Proteins were annotated against the Ciona database and analyzed to predict their molecular functions, roles in biological processes, and position in constructed network pathways. The identified Ciona neural complex proteome was found to map onto vertebrate nervous system pathways, including cytoskeleton remodeling neurofilaments, cell adhesion through the histamine receptor signaling pathway, γ‐aminobutyric acid‐A receptor life cycle neurophysiological process, glycolysis, and amino acid metabolism. The proteome map of the Ciona neural complex is the first step toward a better understanding of several important processes, including the evolution and regeneration capacity of the Ciona nervous system.     

Silk cocoon of Bombyx mori: Proteins and posttranslational modifications – heavy phosphorylation and evidence for lysine‐mediated cross links

Although silk is used to produce textiles and serves as a valuable biomaterial in medicine, information on silk proteins of the cocoon is limited. Scanning electron microscopy was applied to morphologically characterise the sample and the solubility of cocoon in lithium thiocyanate and 2‐DE was carried out with multi‐enzyme in‐gel digestion followed by MS identification of silk‐peptides. High‐sequence coverage of the silk cocoon proteins fibroin light and heavy chain, sericins and fibrohexamerins was revealed and PTMs as heavy phosphorylation of silk fibroin heavy chain; lysine hydroxylation and Lys‐>allysine formation have been observed providing evidence for lysine‐mediated cross linking of silk as found in collagens, which has not been reported so far. Tyrosine oxidation verified the presence of di‐tyrosine cross links. A high degree of sequence conflicts probably representing single‐nucleotide polymorphisms was observed. PTM and sequence conflicts may be modulating structure and physicochemical properties of silk.     

Proteomic analysis of the cold stress response in the moss,Physcomitrella patens

Cold stress has adverse effects on plant growth and development. Plants respond and acclimate to cold stress through various biochemical and physiological processes, thereby acquiring stress tolerance. To better understand the basis for tolerance, we carried out a proteomic study in the model moss, Physcomitrella patens, characterizing gametophore proteins with 2‐DE and mass spectroscopy. Following exposure to 0°C for up to 3 days, out of the more than 1000 protein spots reproducibly resolved, only 45 changed in abundance by at least 1.5‐fold. Of these, 35 were identified by tryptic digestion and mass spectroscopy. Photosynthetic proteins decreased, whereas many catabolic proteins increased. In addition, cold stress up‐regulated a variety of signaling, cytoskeleton, and defense proteins and few proteins in these classes were down‐regulated. Up‐regulated proteins include the 14‐3‐3‐like protein, actin, HSP70s, lipoxygenases, and cytochrome P450 proteins. These results point to pathways that are important for the mechanism of cold stress response in P. patens and by extension to the entire plant kingdom.     

Proteomic analysis of apoptosis related proteins regulated by proto‐oncogene protein DEK

A nuclear phosphoprotein, DEK, is implicated in certain human diseases, such as leukemia and antoimmune disorders, and a major component of metazoan chromatin. Basically as a modulator of chromatin structure, it can involve in various DNA and RNA‐dependent processes and function as either an activator or repressor. Despite of numerous efforts to suggest the biological role of DEK, direct target proteins of DEK in different physiological status remains elusive. To investigate if DEK protein triggers the changes in certain protein networks, DEK was knocked down at both types of cell clones using siRNA expression. Here we provide a catalogue of proteome profiles in total cell lysates derived from normal HeLa and DEK knock‐down HeLa cells and a good in vitro model system for dissecting the protein networks due to this proto‐oncogenic DEK protein. In this biological context, we compared total proteome changes by the combined methods of two‐dimensional gel electrophoresis, quantitative image analysis and MALDI‐TOF MS analysis. There were a large number of targets for DEK, which were differentially expressed in DEK knock‐down cells and consisted of 58 proteins (41 up‐regulated and 17 down‐regulated) differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. In the identified 58 spots, 16% of proteins are known to be associated with apoptosis. Among others, we identified apoptosis related proteins such as Annexins, Enolase1, Lamin A, and Glutathione‐S‐transferase omega 1. These results are consistent with recent studies indicating the crucial role of DEK in apoptosis pathway. We further demonstrated by ChIP analysis that knock‐down of DEK caused hyperacetylation of histones around Prx VI promoter which is upregulated in our profile. Using immunoblotting analysis, we have demonstrated the modulation of other caspase‐dependent apoptosis related proteins by DEK knock‐down and further implicate its role in apoptosis pathway. J. Cell. Biochem. 106: 1048–1059, 2009. © 2009 Wiley‐Liss, Inc.     

Proteome and radioimmunoassay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, β-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.     

Proteome changes in rat plasma in response to sibutramine

Sibutramine is an anti-obesity agent that induces weight loss by selective inhibition of neuronal reuptake of serotonin and norepinephrine; however, it is associated with the risk of cardiovascular diseases (CVD), including heart attack and stroke. Here, we analyzed global protein expression patterns in plasma of control and sibutramine-treated rats using proteomic analysis for a better understanding of the two conflicting functions of this drug, appetite regulation, and cardiovascular risk. The control (n=6) and sibutramine-treated groups (n=6) were injected by vehicle and sibutramine, respectively, and 2-DE combined with MALDI-TOF/MS were performed. Compared to control rats, sibutramine-administered rats gained approximately 18% less body weight and consumed about 13% less food. Plasma leptin and insulin levels also showed a significant decrease in sibutramine-treated rats. As a result of proteomic analysis, 23 differentially regulated proteins were discovered and were reconfirmed by immunoblot analysis. Changed proteins were classified into appetite regulation and cardiovascular risk, according to their regulation pattern. Because the differential levels of proteins that have been well recognized as predictors of CVD risk were not well matched with the results of our proteomic analysis, this study does not conclusively prove that sibutramine has an effect on CVD risk.     

Changes in the proteome and phosphoproteome expression in the bryozoan Bugula neritina larvae in response to the antifouling agent butenolide

Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid‐phase IEF sample prefractionation combined with 2‐DE and MALDI‐TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium‐binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required.     

Proteomic identification of a novel isoform of collapsin response mediator protein-2 in spinal nerves peripheral to dorsal root ganglia

Primary afferent fibers are originated from pseudounipolar sensory cells in dorsal root ganglia (DRG) and transmit external stimuli received in the skin to the spinal cord. Here we undertook a proteomic approach to uncover the polarity of primary afferent fibers. Lumbar spinal nerve segments, peripheral and central to DRG, were dissected from 5-wk-old Wistar rats and the lysates were subjected to large-sized 2-DE at pH 5-6. Among approximately 800 protein spots detected in the central and peripheral fractions, one of the unique spots in the peripheral fraction with MW of 60 kDa and pI of 5.6 was identified as an isoform of collapsin response mediator protein-2 (CRMP-2) by MALDI-TOF MS and Western blots with anti-CRMP-2 antibodies that recognize 1-17 and 486-528 residues. Since this novel spot was detected only in the peripheral fraction, but not in the central fraction, DRG, and other regions of the brain, it was named periCRMP-2. The C-terminal fragment of CRMP-2 was not detected in periCRMP-2 by MS analyses. Expression of periCRMP-2 decreased following sciatic nerve injury. These results suggest that periCRMP-2 is a C-terminal truncated isoform polarized in the peripheral side of spinal nerves and may be involved in nerve degeneration and regeneration.     

Gender differences in rat plasma proteome in response to high-fat diet

Knowledge of gender differences is important because nutritional recommendations on the basis of data collected using predominantly male subjects may not be valid for women. In the present study, we performed proteomic analysis in plasma of rats fed a high-fat diet (HFD) using 2-DE combined with MALDI-TOF-MS for analysis of differential regulation patterns between male and female plasma proteins. Male rats gained more body weight with increased values of biochemical parameters than female rats. Image analysis and further statistical analysis allowed detection and identification of 31 proteins that were significantly modulated in a gender-dependent manner in response to HFD. Those differential expressed proteins were classified into three groups based on their regulation patterns in response to diet and gender. Consequently, we found 13 proteins showing gender-different regulation in both normal diet (ND) and HFD, where 9 proteins showed identical regulation patterns (Group I) and 4 proteins exhibited opposite regulation mode (Group II) between the genders. Eighteen proteins showed no gender-difference but HFD-responsive regulation (Group III). Of these, Apo A-IV, CRP precursor, Hp precursor, and FGG showed a clear gender difference in both ND and HFD, with the same regulation patterns. Present proteomic research into gender-dimorphic protein modulation in plasma would aid in improvement of gender awareness in the health care system and in implementation of evidence-based gender-specific clinical recommendations.     

Proteomic profiling of cerebrospinal fluid identifies prostaglandin D2 synthase as a putative biomarker for pediatric medulloblastoma: A pediatric brain tumor consortium study

The aims of this study were to demonstrate the feasibility of centrally collecting and processing high-quality cerebrospinal fluid (CSF) samples for proteomic studies within a multi-center consortium and to identify putative biomarkers for medulloblastoma in CSF. We used 2-DE to investigate the CSF proteome from 33 children with medulloblastoma and compared it against the CSF proteome from 25 age-matched controls. Protein spots were subsequently identified by a combination of in-gel tryptic digestion and MALDI-TOF TOF MS analysis. On average, 160 protein spots were detected by 2-DE and 76 protein spots corresponding to 25 unique proteins were identified using MALDI-TOF. Levels of prostaglandin D2 synthase (PGD2S) were found to be six-fold decreased in the tumor samples versus control samples (p<0.00001). These data were further validated using ELISA. Close examination of PGD2S spots revealed the presence of complex sialylated carbohydrates at residues Asn(78) and Asn(87) . Total PGD2S levels are reduced six-fold in the CSF of children with medulloblastoma most likely representing a host response to the presence of the tumor. In addition, our results demonstrate the feasibility of performing proteomic studies on CSF samples collected from patients at multiple institutions within the consortium setting.     

MS characterization of qualitative protein polymorphisms in the spinal cords of inbred mouse strains

The spinal cord proteomes of two inbred mouse strains with different susceptibility to experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, were investigated by 2‐DE and MALDI‐MS. A proteome map comprising 304 different protein species was established. Using 2‐D fluorescence difference gel electrophoresis, a comparison of the mouse strains revealed 26 qualitatively polymorphic proteins with altered electrophoretic mobility. MS analyses and DNA sequencing were applied to characterize their structural differences and 14 single amino acid substitutions were identified. Moreover, analysis of selectively enriched phosphopeptides from the neurofilament heavy polypeptide of both mouse strains revealed a high degree of diversity in the phosphorylated C‐terminal domains of this protein. The described approach is capable to structurally characterize qualitative protein polymorphisms, whereas their functional significance remains to be elucidated. For some proteins formerly associated with experimental autoimmune encephalomyelitis and/or multiple sclerosis structural polymorphisms are described here, which may be subjected to further investigations. In addition, this work should be of general interest for proteomic analysis of inbred strains, because it shows potentials and constraints in the use of 2‐DE analysis and MALDI‐MS to detect and characterize structural protein polymorphisms.