High-resolution Two-dimensional Gel Electrophoresis Research Articles

Zn-responsive proteome profiling and time-dependent expression of proteins regulated by MTF-1 in A549 cells.

Zinc plays a critical role in many biological processes. However, it is toxic at high concentrations and its homeostasis is strictly regulated by metal-responsive transcription factor 1 (MTF-1) together with many other proteins to protect cells against metal toxicity and oxidative stresses. In this paper, we used high-resolution two-dimensional gel electrophoresis (2DE) to profile global changes of the whole soluble proteome in human lung adenocarcinoma (A549) cells in response to exogenous zinc treatment for 24 h. Eighteen differentially expressed proteins were identified by MALDI TOF/TOF and MASCOT search. In addition, we used Western blotting and RT-PCR to examine the time-dependent changes in expression of proteins regulated by MTF-1 in response to Zn treatment, including the metal binding protein MT-1, the zinc efflux protein ZnT-1, and the zinc influx regulator ZIP-1. The results indicated that variations in their mRNA and protein levels were consistent with their functions in maintaining the homeostasis of zinc. However, the accumulation of ZIP-1 transcripts was down-regulated while the protein level was up-regulated during the same time period. This may be due to the complex regulatory mechanism of ZIP-1, which is involved in multiple signaling pathways. Maximal changes in protein abundance were observed at 10 h following Zn treatment, but only slight changes in protein or mRNA levels were observed at 24 h, which was the time-point frequently used for 2DE analyses. Therefore, further study of the time-dependent Zn-response of A549 cells would help to understand the dynamic nature of the cellular response to Zn stress. Our findings provide the basis for further study into zinc-regulated cellular signaling pathways.

Topoisomerase 2 is dispensable for the replication and segregation of small yeast artificial chromosomes (YACs).

DNA topoisomerases are thought to play a critical role in transcription, replication and recombination as well as in the condensation and segregation of sister duplexes during cell division. Here, we used high-resolution two-dimensional agarose gel electrophoresis to study the replication intermediates and final products of small circular and linear minichromosomes of Saccharomyces cerevisiae in the presence and absence of DNA topoisomerase 2. The results obtained confirmed that whereas for circular minichromosomes, catenated sister duplexes accumulated in the absence of topoisomerase 2, linear YACs were able to replicate and segregate regardless of this topoisomerase. The patterns of replication intermediates for circular and linear YACs displayed significant differences suggesting that DNA supercoiling might play a key role in the modulation of replication fork progression. Altogether, this data supports the notion that for linear chromosomes the torsional tension generated by transcription and replication dissipates freely throughout the telomeres.

Morphological and proteomic analysis of early stage air-liquid interface biofilm formation in Mycobacterium smegmatis.

We studied the early stages of pellicle formation by Mycobacterium smegmatis on the surface of a liquid medium [air-liquid interface (A-L)]. Using optical and scanning electron microscopy, we showed the formation of a compact biofilm pellicle from micro-colonies over a period of 8-30 h. The cells in the pellicle changed size and cell division pattern during this period. Based on our findings, we created a model of M. smegmatis A-L early pellicle formation showing the coordinate growth of cells in the micro-colonies and in the homogeneous film between them, where the accessibility to oxygen and nutrients is different. A proteomic approach utilizing high-resolution two-dimensional gel electrophoresis, in combination with mass spectrometry-based protein identification, was used to analyse the protein expression profiles of the different morphological stages of the pellicle. The proteins identified formed four expression groups; the most interesting of these groups contained the proteins with highest expression in the biofilm development phase, when the floating micro-colonies containing long and more robust cells associate into flocs and start to form a compact pellicle. The majority of these proteins, including GroEL1, are involved in cell wall synthesis or modification, mostly through the involvement of mycolic acid biosynthesis, and their expression maxima correlated with the changes in cell size and the rigidity of the bacterial cell wall observed by scanning electron microscopy.

A proteomic study on molecular mechanism of poor grain-filling of rice (Oryza sativa L.) inferior spikelets.

Cultivars of rice (Oryza sativa L.), especially of the type with large spikelets, often fail to reach the yield potential as expected due to the poor grain-filling on the later flowering inferior spikelets (in contrast to the earlier-flowering superior spikelets). The present study showed that the size and grain weight of superior spikelets (SS) was greater than those of inferior spikelets (IS), and the carbohydrate supply should not be the major problem for the poor grain-filling because there was adequate amount of sucrose in IS at the initial grain-filling stage. High resolution two-dimensional gel electrophoresis (2-DE) in combination with Coomassie-brilliant blue (CBB) and Pro-Q Diamond phosphoprotein fluorescence stain revealed that 123 proteins in abundance and 43 phosphoproteins generated from phosphorylation were significantly different between SS and IS. These proteins and phosphoproteins were involved in different cellular and metabolic processes with a prominently functional skew toward metabolism and protein synthesis/destination. Expression analyses of the proteins and phosphoproteins associated with different functional categories/subcategories indicated that the starch synthesis, central carbon metabolism, N metabolism and cell growth/division were closely related to the poor grain-filling of IS. Functional and expression pattern studies also suggested that 14-3-3 proteins played important roles in IS poor grain-filling by regulating the activity of starch synthesis enzymes. The proteome and phosphoproteome obtained from this study provided a better understanding of the molecular mechanism of the IS poor grain-filling. They were also expected to be highly useful for improving the grain filling of rice.

New insight into the castrated mouse epididymis based on comparative proteomics

The mammalian epididymis is an important male accessory gland where the spermatozoa gain the ability to fertilise the egg. To further understand the effects of testicular factors on the epididymis, the proteome of castrated adult mice and sham controls was compared using high-resolution two-dimensional gel electrophoresis following identification of proteins by matrix-assisted laser desorption ionisation time-of-flight/time-of-flight mass spectrometry. Twenty-three differentially expressed proteins (11 upregulated and 12 downregulated) were identified in epididymides from castrated. Bioinformatic analysis indicated that these castration-responsive proteins participated in energy metabolism and the antigen processing and presentation pathway. The differential expression levels were further validated by western blotting. The differentially expressed proteins may serve as potential candidates in studies of epididymal function and male infertility.

A Review on Study and Comparison between 2D Gel Electrophoresis and Mass Spectrometry

Protein structure prediction is an important area of life sciences. The Protein prophecy is unbreakable because of its complex structure. The main problem in proteomics is to estimate the several proteins available in cell structure or tissue sample. The protein-protein interaction is essential to study the behaviour of protein in a specific environment, and study molecular relationship in living systems. Therefore, large scale proteomics technologies are required to measure physical connection of proteins in living organisms. A combination of high resolution two-dimensional (2-D)gel electrophoresis, highly sensitive biological mass spectrometry , and the speedily growing protein and DNA databases has resulted for high throughput proteomics. This review concentrates on two main techniques of protein identification 2-D gel electrophoresis and High Throughput Mass Spectrometry.

2D-DIGE proteome analysis on the platelet proteins of patients with major depression

IntroductionPlatelet activation is related to the psychopathology of major depression. We attempted to search and identify protein biomarkers from the platelets of patients with major depression. High resolution two-dimensional Differential Gel Electrophoresis (2D-DIGE), the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), Western blot, and bioinformatic tools were applied to examine the platelet proteins of 10 patients with major depression and 10 healthy controls.ResultsThe levels of 8 proteins were significantly different between the patients with major depression in the acute phase and healthy controls. The levels of protein disulfide-isomerase A3 (PDIA3) and F-actin-capping protein subunit beta (CAPZB) were higher in patients with major depression than in healthy controls. The levels of fibrinogen beta chain (FIBB), fibrinogen gamma chain (FIBG), retinoic acid receptor beta (RARB), glutathione peroxidase 1 (GPX1), SH3 domain-containing protein 19 (SH319), and T-complex protein 1 subunit beta (TCPB) were lower in patients with major depression than in healthy controls.ConclusionsPlatelet provided valuable information about the pathways and processes of inflammation/immunity, oxidative stress, and neurogenesis, related to major depression.

Proteomic Analysis of Cucumber Defense Rresponses Induced by Propamocarb

Propamocarb is an agricultural chemical that has been widely used to protect cucumber plants from downy mildew. To understand the mechanisms of cucumber defense responses to propamocarb, we investigated the physiological and proteomic responses of the cucumber line D0351 with propamocarb application. We found that after treatment with propamocarb, the activities of detoxifying enzymes (glutathione reductase, GR; glutathione S-tramsferase, GST) and soluble sugar content of cucumber fruit were significantly increased, but malonaldehyde (MDA) content was significantly reduced. To identify components of propamocarb responsive signaling, we compared the high resolution two-dimensional gel electrophoresis (2-DE) protein profiles of control and propamocarb-treated fruits, and identified 18 differentially expressed (13 up-regulated and 5 down-regulated) proteins induced by propamocarb which were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The majority of the proteins had functions related to detoxication, energy and transport, protein biosynthesis, regulating reactions and defending against stresses. A real-time quantitative reverse transcriptional-polymerase chain reaction (qRT-PCR) was used to compare transcript and protein accumulation patterns for 18 candidate proteins, and the expression of 14 was consistent at both transcript and protein levels. The responses of cucumber proteome to propamocarb seemed complex; the identified proteins may play an important role in regulating adaptation activities following exposure to propamocarb. Data presented herein may shed light on understanding cucumber fruit defense responses under propamocarb treatment.

Soluble Alpha-APP (sAPPalpha) Regulates CDK5 Expression and Activity in Neurons

A growing body of evidence suggests a role for soluble alpha-amyloid precursor protein (sAPPalpha) in pathomechanisms of Alzheimer disease (AD). This cleavage product of APP was identified to have neurotrophic properties. However, it remained enigmatic what proteins, targeted by sAPPalpha, might be involved in such neuroprotective actions. Here, we used high-resolution two-dimensional polyacrylamide gel electrophoresis to analyze proteome changes downstream of sAPPalpha in neurons. We present evidence that sAPPalpha regulates expression and activity of CDK5, a kinase that plays an important role in AD pathology. We also identified the cytoprotective chaperone ORP150 to be induced by sAPPalpha as part of this protective response. Finally, we present functional evidence that the sAPPalpha receptor SORLA is essential to mediate such molecular functions of sAPPalpha in neurons.

An integrated proteomic approach to decipher the effect of methyl jasmonate elicitation on the proteome of Silybum marianum L. hairy roots

Jasmonate and its methyl derivative, methyl jasmonate (MeJA), are naturally occurring compounds that mediate several plant physiological processes in response to pathogen attack, wounding, and ozone. Exogenous application of jasmonates triggers defense responses that resemble those initiated by pathogen infection and also modulates the production of certain secondary metabolites in a variety of plant species. In this study, we treated the hairy root cultures of Silybum marianum L. with 100μM MeJA and then measured the content of Silymarin (SLM). We observed that the SLM content increased significantly after 48h of MeJA treatment and remained constant for 120h. However, MeJA treatment caused a significant growth reduction after 96h incubation. The activity of lipoxygenase as a key enzyme in the jasmonate biosynthesis pathway and anti-oxidative enzymes; peroxidase and ascorbate peroxidase was also significantly increased after MeJA treatment. To elucidate the global effect of jasmonate on gene expression of S.marianum, we employed high resolution two-dimensional gel electrophoresis coupled with tandem mass spectrometry. Out of 670 reproducibly detected protein spots which were analyzed on each given gel, 32 spots were up- or down regulated upon MeJA treatment. Of them, ten proteins such as ER binding protein, glutamine synthetase, pathogenesis-related protein, caffeoyl CoA O-methyltransferase, and profilin-1 could be identified by mass spectrometry analysis. The possible implications of the identified proteins on physiological outcome of MeJA application in S.marianum hairy root culture will be discussed.

Monitoring post mortem changes in porcine muscle through 2-D DIGE proteome analysis of Longissimus muscle exudate

BackgroundMeat quality is a complex trait influenced by a range of factors with post mortem biochemical processes highly influential in defining ultimate quality. High resolution two-dimensional DIfference Gel Electrophoresis (2-D DIGE) and Western blot were applied to study the influence of post mortem meat ageing on the proteome of pork muscle. Exudate collected from the muscle following centrifugation was analysed at three timepoints representing a seven day meat ageing period.ResultsThe intensity of 136 spots varied significantly (p < 0.05) across this post mortem period and 40 spots were identified using mass spectrometry. The main functional categories represented were metabolic proteins, stress-related proteins, transport and structural proteins. Metabolic and structural proteins were generally observed to increase in abundance post mortem and many likely represent the accumulation of the degradation products of proteolytic enzyme activity. In contrast, stress-related proteins broadly decreased in abundance across the ageing period. Stress response proteins have protective roles in maintaining cellular integrity and a decline in their abundance over time may correlate with a reduction in cellular integrity and the onset of meat ageing. Since cellular conditions alter with muscle ageing, changes in solubility may also contribute to observed abundance profiles.ConclusionsMuscle exudate provided valuable information about the pathways and processes underlying the post mortem ageing period, highlighting the importance of post mortem modification of proteins and their interaction for the development of meat quality traits.

Ascaris lumbricoides and Ascaris suum: Comparative proteomic studies using 2-DE coupled with mass spectrometry

Roundworms of the genus Ascaris comprise species of medical (A. lumbricoides) and veterinary (A. suum) importance. Despite the economic importance, public health impact, and worldwide prevalence both parasites still have a controversial taxonomy and their proteomes are poorly described in databases. In the present study, high-resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF MS was used to characterize and contrast proteomic profiles of adult female A. lumbricoides and A. suum. Approximately, 3-fold higher protein spots were obtained in the present study compared to traditional method, and more than 630 and 750 protein spots were reproducibly detected among the 6 gels examined (3 gels for each of A. lumbricoides and A. suum). The peptide mass finger printings (PMFs) were acquired successfully for 17 from 22 differential 2-DE spots, corresponding to 12 unique proteins. Among them, 2 Asu-enriched and 3 Alu-specific proteins were found in A. lumbricoides; and 6 Asu-enriched and 1 Alu-specific proteins were found in A. suum. Functional analysis revealed low level of difference among differentially expressed proteins of the two nematode species, while the 1 species-specific protein of A. suum was different from that of A. lumbricoides. The proteomic patterns of A. lumbricoides and A. suum provide proteomic evidence that these two taxa are very closely related. This work indicates that the combined 2-DE and MALDI-TOF/TOF MS approaches employed in the current study allow the identification of proteins whose expression is different between Ascaris species.

On-Membrane Digestion Technology for Muscle Proteomics

High-resolution two-dimensional gel electrophoresis and in-gel digestion are routinely used for large-scale protein separation and peptide generation in mass spectrometry-based proteomics, respectively. However, the combination of isoelectric focusing in the first dimension and polyacrylamide slab gel electrophoresis in the second dimension is not suitable for the proper separation of integral proteins and high-molecular-mass proteins. In addition, in-gel trypsination may not result in a high degree of efficient digestion levels for the production of large numbers of peptides in the case of certain protein species. The application of gradient one-dimensional gel electrophoresis and on-membrane digestion can overcome these technical problems and be extremely helpful for the comprehensive identification of proteins that are underrepresented in routine two-dimensional gel electrophoretic approaches. This review critically examines the general application of on-membrane digestion techniques in proteomics and its recent application for the identification of very large integral membrane proteins from skeletal muscle by mass spectrometry. This includes the discussion of proteomic studies that have focused on the proteomic characterization of the membrane cytoskeletal protein dystrophin from sarcolemma vesicles and the ryanodine receptor calcium release channel of the sarcoplasmic reticulum from skeletal muscle.

An improved plant leaf protein extraction method for high resolution two-dimensional polyacrylamide gel electrophoresis and comparative proteomics

We report here a simple and universally applicable protocol for extracting high quality proteins from plant leaf tissues. The protocol provides improved resolution and reproducibility of two-dimensional polyacrylamide gel electrophoresis (2-DE) and reduces the time required to analyze samples. Partitioning rubisco by polyethylene glycol (PEG) fractionation provides clearer detection of low-abundance proteins. Co-extraction of interfering substances increases the sample conductivity, which results in poor electrophoretic separation. Re-extraction of PEG-fractionated samples with phenol effectively eliminated interfering substances, which results in optimal conductivity during separation in the first dimension of the isoelectric focusing. Smooth focusing reduces analysis time and provides superior resolution in 2-DE gels. Incubating the samples at −80° C instead of −20° C reduced protein precipitation time to 2–3 h. Removal of nonprotein contaminants and the use of sonication increased protein solubility without additional reagents. These changes enabled loading and separation of maximum amounts of proteins, which permitted improved protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). An immunological approach revealed that little or no ribulose-1, 5-bisphosphte bisphosphate carboxylase oxygenase was present in the PEG supernatant. In addition, low-abundance proteins, such as myelocytomatosis transcription factor (MYC) and alpha subunit of heterotrimeric guanine nucleotide-binding protein complex (Gα), were detected only in the modified PEG supernatant and not in the total protein. These results suggest that our protocol produced high quality proteins and made many low-abundant proteins available for proteomic analysis. The successful application of this protocol for analyzing the leaf proteomes of soybean, Miscanthus sinensis, barley, Chinese cabbage, peanut and tea (Camellia sinensis) suggests that it could be used for comparative proteomic analysis of a wide range of plant leaves.

Proteomic characterization of a novel structural protein ORF5a of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) emerged in the late 1980s in both Europe and North America is an arterivirus causing great economic losses in the pig industry worldwide. Recently, a novel structural protein, ORF5a, encoded by an alternative ORF of a subgenomic mRNA encoding the major envelope glycoprotein, GP5, has been discovered in all arteriviruses, suggesting its important role in arterivirology. The present study attempted to investigate compensatory changes of cellular gene expression in natural target cells regulated by the ORF5a. We thus established sublines of PAM cells to stably express the PRRSV ORF5a protein and assessed alterations in cellular protein productions of ORF5a-expressing PAM (PAM-ORF5a) cells at different time courses by the use of proteomic analysis. A total of 36 protein spots were initially found to be differentially expressed in PAM-ORF5a cells compared with normal PAM cells by high-resolution two-dimensional gel electrophoresis (2DE). Of these spots, 16 protein spots with statistically significant alteration, including 13 up-regulated and 3 down-regulated protein spots, were picked out for subsequent protein identification by peptide mass fingerprinting after matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS). The altered cellular proteins identified in this study were grouped into the functions associated with a variety of cellular processes such as cell growth, cytoskeleton networks and cell communication, metabolism, protein biosynthesis, RNA processing, and transportation. The proteomics data will provide valuable information for better understanding the specific cellular response to the novel ORF5a protein during PRRSV replication.

Down-Regulation of Small Rubber Particle Protein Expression Affects Integrity of Rubber Particles and Rubber Content in Taraxacum brevicorniculatum

The biosynthesis of rubber is thought to take place on the surface of rubber particles in laticifers, highly specialized cells that are present in more than 40 plant families. The small rubber particle protein (SRPP) has been supposed to be involved in rubber biosynthesis, and recently five SRPPs (TbSRPP1–5) were identified in the rubber-producing dandelion species Taraxacum brevicorniculatum. Here, we demonstrate by immunogold labeling that TbSRPPs are localized to rubber particles, and that rubber particles mainly consist of TbSRPP3, 4 and 5 as shown by high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis. We also carried out an RNA-interference approach in transgenic plants to address the function of TbSRPPs in rubber biosynthesis as well as rubber particle morphology and stability. TbSRPP-RNAi transgenic T. brevicorniculatum plants showed a 40–50% reduction in the dry rubber content, but neither the rubber weight average molecular mass nor the polydispersity of the rubber were affected. Although no phenotypical differences to wild-type particles could be observed in vivo, rubber particles from the TbSRPP-RNAi transgenic lines were less stable and tend to rapidly aggregate in expelling latex after wounding of laticifers. Our results prove that TbSRPPs are very crucial for rubber production in T. brevicorniculatum, probably by contributing to a most favourable and stable rubber particle architecture for efficient rubber biosynthesis and eventually storage.

A discovery-phase urine proteomics investigation in type 1 diabetes

Diabetes is a chronic metabolic disease which can lead to serious health problems particularly in and to the development of cardiovascular and renal complications. The aim of this study is to possibly identify distinctive molecular features in urine samples which might correlate to the progression and complications of type 1 diabetes. Diabetic patients with normo- and micro-albuminuria have been analyzed and compared to a group of control subjects. Urine proteins of control and type 1 diabetes subjects were investigated in their proteome profiles, using high-resolution two-dimensional gel electrophoresis separation and protein identifications by MALDI-TOF-MS and LC-MS/MS analysis. Proteomics analysis highlighted differential expression of several proteins between control and type 1 diabetes subjects. In particular, five proteins were found to be down-regulated and four proteins up-regulated. Lower protein representations in diabetic subjects were associated with Tamm-Horsfall urinary glycoprotein, apolipoprotein A-I, apolipoprotein E, α2-thiol proteinase inhibitor, and human complement regulatory protein CD59, while higher protein representations were found for α-1-microglobulin, zinc-α2 glycoprotein, α-1B glycoprotein, and retinol-binding protein 4. These differences were maintained comparing control subjects with type 1 diabetes normo-albuminuric and micro-albuminuric subjects. Furthermore, these proteins are correlated to glycosylated hemoglobin and microalbuminuria, confirming their role in diabetic pathology. This study gives new insights on potential molecular mechanisms associated with the complications of type 1 diabetic disease providing evidences of urine proteins potentially exploitable as putative prognostic biomarkers.

Proteins differentially expressed in elicited cell suspension culture of Podophyllum hexandrum with enhanced podophyllotoxin content.

BackgroundPodophyllotoxin (PTOX), the precursor for semi-synthesis of cancer therapeutics like etoposide, teniposide and etophos, is primarily obtained from an endangered medicinal herb, Podophyllum hexandrum Royle. PTOX, a lignan is biosynthetically derived from the phenylpropanoid pathway. The aim of this study is to investigate changes in the P. hexandrum cell proteome potentially related to PTOX accumulation in response to methyl jasmonate (MeJA) elicitation. High-resolution two-dimensional gel electrophoresis (2-DE) followed by colloidal Coomassie staining and mass spectrometric analysis was used to detect statistically significant changes in cell’s proteome.ResultThe HPLC analysis showed approximately 7–8 fold change in accumulation of PTOX, in the 12day old cell suspension culture (i.e. after 9days of elicitation) elicited with 100 μM MeJA as compared to the control. Using 2-DE a total of 233 spots was detected, out of which 105 spots were identified by MALDI TOF-TOF MS/MS. Data were subjected to functional annotation from a biological point of view through KEGG. The phenylpropanoid and monolignol pathway enzymes were identified, amongst these, chalcone synthase, polyphenol oxidase, caffeoyl CoA 3-O-methyltransferase, S-adenosyl-L-methionine-dependent methyltransferases, caffeic acid-O-methyl transferase etc. are noted as important. The relation of other differentially accumulated proteins with varied effects caused by elicitors on P. hexandrum cells namely stress and defense related protein, transcription and DNA replication and signaling are also discussed.ConclusionsElicitor-induced PTOX accumulation in P. hexandrum cell cultures provides a responsive model system to profile modulations in proteins related to phenylpropanoid/monolignol biosynthesis and other defense responses. Present findings form a baseline for future investigation on a non-sequenced medicinal herb P. hexandrum at molecular level.

Prognostic value of nuclear matrix protein expression in localized prostate cancer

The aim of the study was to correlate nuclear matrix (NM) protein expression profiles with the risk of PSA progression or death in early prostate cancer (PCa). High-resolution two-dimensional gel electrophoresis (2D-PAGE) was used to identify tumor-associated NM proteins in the PCa specimens obtained from 94 patients. The association between the expression of each protein and the probability of PSA progression or death was studied through univariate analysis. Unsupervised hierarchical clustering analysis was then used to generate patient clusters showing comparable outcomes by including the proteins that were predictive at univariate analysis. PSA-free and overall survival curves relative to each cluster were constructed by means of the Kaplan-Meier method and curves compared by the log-rank test. Multi-parametric models were constructed according to Cox proportional hazard technique. After a median follow-up of 11.7 years (range, 6.5-16.2), 50 patients progressed and 22 died. Of the eight NM proteins identified through 2D-PAGE, proteins NM-6, NM-7 and NM-8 were confirmed to be individually associated with a higher risk of PSA progression at univariate analysis. Proteins NM-6 and NM-8 were also predictive of survival probability. Hierarchical clustering analysis of these proteins allowed to identify one cluster of tumors co-expressing the three proteins or proteins NM-6 and NM-8, characterized by a very poor outcome, suggesting a specific role for these proteins in PCa progression. The predictive value of this mini-signature in respect to PSA-free survival was confirmed by multivariate analysis. Changes in NM scaffolding are strongly associated with the clinical outcome of patients following radical prostatectomy.

Identification of specific nuclear structural protein alterations in human breast cancer

Breast cancer is the most commonly diagnosed type of cancer and a major cause of death in women. Reliable biomarkers are urgently needed to improve early detection or to provide evidence of the prognosis for each individual patient through expression levels in tumor tissue or body fluids. This proteomic analysis focused on the nuclear structure of human breast cancer tissue, which has been shown to be a promising tool for cancer biomarker development. The nuclear matrix composition of human breast cancer (n = 14), benign controls (n = 2), and healthy controls (n = 2) was analyzed by high-resolution two-dimensional gel electrophoresis and mass spectrometry. Validation studies were performed in an individual sample set consisting of additional breast cancer tissues (n = 3) and additional healthy control tissues (n = 2) by one-dimensional immunoblot. In this setting, we identified five proteins that were upregulated in human breast cancer tissue, but absent in the healthy and benign controls (P < 0.001). These spots were also present in the investigated human breast cancer cell lines, but absent in the MCF10a cell line, which represents normal human epithelial breast cells. Two of the breast cancer-specific proteins have been confirmed to be calponin h2 and calmodulin-like protein 5 by one-dimensional immunoblot. This is the first study demonstrating the expression of both proteins in human breast cancer tissue. Further studies are required to investigate the potential role of these proteins as biomarkers for early diagnosis or prognosis in human breast cancer.