Two-dimensional Difference Gel Electrophoresis Research Articles

DIGE Analysis of Animal Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation and accurate within-gel matching. DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometry techniques and further analyzed by other biochemical methods. In this chapter, we describe the application of this methodology to separation and quantitation of proteins mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in the meat industry.

DIGE-Based Phosphoproteomic Analysis.

Here, we describe the detailed step-by-step protocol for detection of phosphoproteins in two-dimensional difference gel electrophoresis (DIGE) gels. A standard DIGE protocol is combined with subsequent post-staining with phosphospecific fluorescent dye. The combination of these two methods complements DIGE-based proteome profiling by fluorescence detection of phosphoproteins in the same gel providing additional possibility for sensitive and accurate quantification of the differentially regulated phosphoproteins in biological samples.

DIGE Analysis of Fish Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) appears to be especially useful in quantitative approaches, allowing the co-separation of proteins of control samples from proteins of treatment/disease samples on the same gel, eliminating gel-to-gel variability. The principle of 2D-DIGE is to label proteins prior to isoelectric focusing and use three spectrally resolvable fluorescent dyes, allowing the independent labeling of control and experimental samples. This procedure makes it possible to reduce the number of gels in an experiment, allowing the accurate and reproducible quantification of multiple samples. 2D-DIGE has been found to be an excellent methodical tool in several areas of fish research, including environmental pollution and toxicology, the mechanisms of development and disorders, reproduction, nutrition, evolution, and ecology.

Subcellular Fractionation for DIGE-Based Proteomics.

Mass spectrometry-based protein methodologies have revolutionized the field of analytical biochemistry and enable the identification of hundreds to thousands of proteins in biological fluids, cell lines, and tissue. This methodology requires the initial separation of a protein constellation and this has been successfully achieved using gel-based techniques, particularly that of two-dimensional difference gel electrophoresis (2D-DIGE). However, given the complexity of the proteome, fractionation techniques may be required to optimize the detection of low-abundance proteins, which are often under-represented, but which may represent important players in health and disease. Such subcellular fractionation protocols typically utilize density-gradient centrifugation and have enabled the enrichment of crude microsomes, the cytosol, the plasmalemma, the nuclei, and the mitochondria. In this chapter, we describe the experimental steps involved in the enrichment of crude microsomes from skeletal muscle using differential centrifugation and subsequent verification of enrichment by gel electrophoresis and immunoblotting, prior to comparative DIGE analysis.

Integrated physiological and proteomic analysis reveals underlying response and defense mechanisms of Brachypodium distachyon seedling leaves under osmotic stress, cadmium and their combined stresses

Drought stress, a major abiotic stress, commonly occurs in metal-contaminated environments and affects crop growth and yield. In this study, we performed the first integrated phenotypic, physiological, and proteomic analysis of Brachypodium distachyon L. seedling leaves under polyethylene glycol (PEG) mock osmotic stress, cadmium (Cd2+), and their combined stresses. Combined osmotic and Cd2+ stress had more significant effects than each individual stress on seedling growth, and the physiological traits and ultrastructures of leaves. Totally 117 differentially accumulated protein (DAP) spots detected by two-dimensional difference gel electrophoresis (2D-DIGE) were identified, and representing 89 unique proteins under individual and combined stresses. These DAPs were involved in photosynthesis/respiration (34%), energy and carbon metabolism (21%), stress/defense/detoxification (13%), protein folding and degradation (12%), and amino acid metabolism (7%). Principal component analysis (PCA) revealed that DAPs from the Cd2+ and combined stresses grouped much closer than those from osmotic stress, indicating Cd2+ and combined stresses resulted in more changes to the leaf proteome than osmotic stress alone. Protein-protein interaction analyses showed that a 14-3-3 centered sub-network could play important roles in responses to abiotic stresses. An overview pathway of proteome metabolic changes in Bd21 seedling leaves under combined stresses is proposed, representing a synergistic responsive network and underlying response and defense mechanisms. SignificanceDrought stress is one of the major abiotic stresses, which commonly occurs in metal-contaminated environments, and affects crop growth and yield performance. We performed the first integrated phenotypic, physiological and proteomic analysis of Brachypodium distachyon L. seedling leaves under drought (PEG), cadmium (Cd2+) and their combined stresses.

Proteomic Analysis of Thermal Regulation of Small Yellow Follicles in Broiler-Type Taiwan Country Chickens.

Heat stress hampers egg production and lowers fertility in layers. This study investigated global protein abundance in the small yellow follicles (SYFs, 6–8 mm diameter) of a broiler-type strain of Taiwan country chickens (TCCs) under acute heat stress. Twelve 30-week-old TCC hens were allocated to a control group maintained at 25°C, and to three acute heat-stressed groups subjected to 38°C for 2 h without recovery, with 2-h recovery, or with 6-h recovery. Two-dimensional difference gel electrophoresis analysis identified 119 significantly differentially expressed proteins after acute heat exposure. Gene ontology analysis revealed that most of these proteins are involved in molecular binding (34%), catalytic activity (23%), and structural molecule activity (11%), and participate in metabolic processes (20%), cellular processes (20%), and cellular component organization or biogenesis (11%). Proteins associated with stress response and survival (HSP25, HSP47, HSP70, HSC70, HSPA9), cytoskeleton remodeling, mitochondrial metabolic process of ATP production, antioxidative defense (peroxiredoxin-6), cargo lipid export and delivery (vitellogenin, apolipoprotein B and A1), and toxin/metabolite clearance and delivery (albumin) were upregulated after acute heat stress in the SYFs of TCCs. No overt cell death and atresia were observed in SYFs after acute heat stress. Collectively, these responses may represent a protective mechanism to maintain follicle cell integrity and survival, thereby ensuring a sufficient pool of SYFs for selection into the ovulation hierarchy for successful egg production.

Pathological features of primary sclerosing cholangitis identified by bile proteomic analysis

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown origin. Previous bile proteomic analyses in patients with PSC have revealed changes in disease activity specific to malignant transformation. In this study, we established a reference bile duct-derived bile proteome for PSC that can be used to evaluate biliary pathophysiology. Samples were collected from patients with PSC or with choledocholithiasis (control) (n=6 each). Furthermore, patients with PSC-associated cholangiocarcinoma (CC) and with CC without concomitant PSC were analyzed. None of the patients showed signs of inflammation or infection based on clinical and laboratory examinations. Proteins overexpressed in patients with PSC relative to control patients were detected by two-dimensional difference gel electrophoresis and identified by liquid chromatography-tandem mass spectrometry. Functional proteomic analysis was performed using STRING software. A total of 101 proteins were overexpressed in the bile fluid of patients with PSC but not in those of controls; the majority of these were predicted to be intracellular and related to the ribosomal and proteasomal pathways. On the other hand, 91 proteins were found only in the bile fluid of controls; most were derived from the extracellular space and were linked to cell adhesion, the complement system, and the coagulation cascade. In addition, proteins associated with inflammation and the innate immune response—e.g., cluster of differentiation 14, annexin-2, and components of the complement system—were upregulated in PSC. The most prominent pathways in PSC/CC-patients were inflammation associated cytokine and chemokine pathways, whereas in CC-patients the Wnt signaling pathway was upregulated. In PSC/CC-patients DIGE-analysis revealed biliary CD14 and Annexin-4 expression, among others, as the most prominent protein that discriminates between both cohorts.Thus, the bile-duct bile proteome of patients with PSC shows disease-specific changes associated with inflammation and the innate immune response even in the absence of obvious clinical signs of cholangitis, malignancy, or inflammation. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.

Evaluation of Beauveria bassiana infection in thehemolymph serum proteins of the housefly, Musca domestica L. (Diptera: Muscidae).

Beauveria bassiana plays a prominent role in biocontrol of houseflies, Musca domestica (L.). Thus, a deeper insight into immune response of M. domestica during B. bassiana infection was warranted to assist the production of more efficient mycoinsecticides. The present study investigates changes in protein profile of M. domestica hemolymph serum post B. bassiana infection using two-dimensional difference gel electrophoresis (2D-DIGE) followed by identification of selected proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The non-infected or control group of flies showed an expression of 54 proteins, while M. domestica infected with B. bassiana expressed a total of 68 hemolymph serum proteins. Thirty three proteins were expressed in both groups of houseflies, whereas 35 proteins were exclusively expressed in infected flies and 21 proteins were exclusively expressed in control flies. Among the 33 proteins which were expressed in both groups of houseflies, 17 proteins showed downregulation, while16 proteins were upregulated in the infected flies compared to the non-infected ones. The results from this study are expected to facilitate better understanding of insect's immune response mechanism.

Preparation of intracellular proteins from a white-rot fungus surrounded by polysaccharide sheath and optimization of their two-dimensional electrophoresis for proteomic studies

The functions and properties of fungal sheath, an extracellular polysaccharide produced by many white-rot fungi, have been studied. However, the strong adherence of the sheath to fungal hyphae had been a major impediment in preparing intracellular proteins from the fungi and analyzing their cellular responses. To overcome this issue, we developed a rapid and easy method to remove the polysaccharide sheath using a selective lignin degrader, Ceriporiopsis subvermispora, which produces large sheath amounts in the presence of a lignin-derived aromatic compound. Using this approach, we achieved thorough removal of sheath and cell disruption using beads and a solution with a high protein-solubilizing power, which enabled the efficient extraction of intracellular proteins from C. subvermispora surrounded by sheath. In addition, for proteomic analysis, we investigated whether these extracted proteins were compatible with two-dimensional electrophoresis. By efficiently concentrating on protein solubilization in the first dimension and using a stacking gel in the second dimension, we successfully obtained a high-resolution proteome map of C. subvermispora. We also used the same proteins for fluorescence two-dimensional difference gel electrophoresis to obtain the quantitative protein expression profiles. These steps demonstrated that two-dimensional electrophoresis-based proteomics can be used to clarify the composition of intracellular proteins from sheath-producing white-rot fungi.

Potential molecular mechanisms for fruiting body formation of Cordyceps illustrated in the case of Cordyceps sinensis

ABSTRACTThe fruiting body formation mechanisms of Cordyceps sinensis are still unclear. To explore the mechanisms, proteins potentially related to the fruiting body formation, proteins from fruiting bodies, and mycelia of Cordyceps species were assessed by using two-dimensional fluorescence difference gel electrophoresis, and the differential expression proteins were identified by matrix-assisted laser desorption/ionisation tandem time of flight mass spectrometry. The results showed that 198 differential expression proteins (252 protein spots) were identified during the fruiting body formation of Cordyceps species, and 24 of them involved in fruiting body development in both C. sinensis and other microorganisms. Especially, enolase and malate dehydrogenase were first found to play an important role in fruiting body development in macro-fungus. The results implied that cAMP signal pathway involved in fruiting body development of C. sinensis, meanwhile glycometabolism, protein metabolism, energy metabolism, and cell reconstruction were more active during fruiting body development. It has become evident that fruiting body formation of C. sinensis is a highly complex differentiation process and requires precise integration of a number of fundamental biological processes. Although the fruiting body formation mechanisms for all these activities remain to be further elucidated, the possible mechanism provides insights into the culture of C. sinensis.

Proteomic profiling of the antifungal drug response of Aspergillus fumigatus to voriconazole

Antifungal resistance is an emerging problem and one of the reasons for treatment failure of invasive aspergillosis (IA). Voriconazole has become a standard therapeutic for the treatment of this often fatal infection. We studied the differentially expressed proteins as a response of Aspergillus fumigatus to voriconazole by employing the two-dimensional difference gel electrophoresis (DIGE) technique. Due to addition of drug, a total of 135 differentially synthesized proteins were identified by MALDI-TOF/TOF-mass spectrometry. In particular, the level of proteins involved in the general stress response and cell detoxification increased prominently. In contrast, cell metabolism and energy proteins were down-regulated, which suggests the cellular effort to maintain balance in energy utilization while trying to combat the cellular stress exerted by the drug. We detected several so-far uncharacterized proteins which may play a role in stress response and drug metabolism and which could be future targets for antifungal treatment. A mutant strain, which is deleted in the cross-pathway control gene cpcA, was treated with voriconazole to investigate the contribution of the general control of amino acid biosynthesis to drug resistance. We compared the mutant strain’s protein expression profile with the wild-type strain. The absence of CpcA led to an increased resistance to voriconazole and a reduced activation of some general stress response proteins, while the transcript level of the triazole target gene erg11A (cyp51A) remained unchanged. In contrast, the sensitivity of strain ΔcpcA to terbinafine and amphotericin B was slightly increased. These findings imply a role of CpcA in the cellular stress response to azole drugs at the post transcriptional level.

Differential Proteomic Analysis of Gender-dependent Hepatic Tumorigenesis in Hras12V Transgenic Mice

Male prevalence is an outstanding characteristic of hepatocellular carcinoma (HCC), and the underlying mechanisms for this have remained largely unknown. In the present study, Hras12V transgenic mice, in which hepatocyte-specific expression of the ras oncogene induces male-biased hepatic tumorigenesis, were studied, and altered proteins were detected by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Protein samples from hepatic tumor tissues (T) and peritumor tissues (P) of transgenic males and females and the corresponding normal liver tissues (Wt) of nontransgenic males and females were subjected to pairwise comparisons based on proteomic analysis. Among 2381 autodetected protein spots, more than 1600 were differentially expressed based on a pairwise comparison (|ratio| > = 1.5, p < = 0.05). Of these, 180 spots were randomly selected for matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) identification; finally, 89 distinct proteins were obtained. Among these 89 proteins, 7 and 50 proteins were further validated by Western blotting and literature investigation, respectively. Intriguingly, compared with Wt, the altered proteins were relatively concentrated in T in transgenic females but in P in transgenic males. Consistently, the levels of p-ERK and p-mTOR were significantly higher in the T of females compared with that of males. The pathway enrichment assay showed that 5 pathways in males but only 1 in females were significantly altered in terms of the upregulated proteins in T compared with Wt. These data indicate that female hepatocytes are disturbed by oncogenes with great difficulty, whereas male hepatocytes readily do so. In addition, 33 proteins were gender-dependently altered in hepatic tumorigenesis. Moreover, 4% DNA packaging and 4% homeostasis-related functional proteins were found in females but not in males, and more nucleus proteins were found in females (8%) than in males (3%). In conclusion, the proteomic data and comparative analysis presented here offer crucial clues for elucidating the mechanisms that underlie the male prevalence in HCC.

Differential protein expression profile in the hypothalamic GT1-7 cell line after exposure to anabolic androgenic steroids.

The abuse of anabolic androgenic steroids (AAS) has been considered a major public health problem during decades. Supraphysiological doses of AAS may lead to a variety of neuroendocrine problems. Precisely, the hypothalamic-pituitary-gonadal (HPG) axis is one of the body systems that is mainly influenced by steroidal hormones. Fluctuations of the hormonal milieu result in alterations of reproductive function, which are made through changes in hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH). In fact, previous studies have shown that AAS modulate the activity of these neurons through steroid-sensitive afferents. To increase knowledge about the cellular mechanisms induced by AAS in GnRH neurons, we performed proteomic analyses of the murine hypothalamic GT1-7 cell line after exposure to 17α-methyltestosterone (17α-meT; 1 μM). These cells represent a good model for studying regulatory processes because they exhibit the typical characteristics of GnRH neurons, and respond to compounds that modulate GnRH in vivo. Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry analyses identified a total of 17 different proteins that were significantly affected by supraphysiological levels of AAS. Furthermore, pathway analyses showed that modulated proteins were mainly associated to glucose metabolism, drug detoxification, stress response and cell cycle. Validation of many of these proteins, such as GSTM1, ERH, GAPDH, PEBP1 and PDIA6, were confirmed by western blotting. We further demonstrated that AAS exposure decreased expression of estrogen receptors and GnRH, while two important signaling pathway proteins p-ERK, and p-p38, were modulated. Our results suggest that steroids have the capacity to directly affect the neuroendocrine system by modulating key cellular processes for the control of reproductive function.

The effect of Fusarium culmorum infection and deoxynivalenol (DON) application on proteome response in barley cultivars Chevron and Pedant

Fusarium head blight (FHB) disease adversely affects grain quality and final yield in small-grain cereals including barley. In the present study, the effect of an artificial infection with Fusarium culmorum and an application of deoxynivalenol (DON) on barley spikes of cultivars Chevron and Pedant during flowering was investigated at grain mid-dough stage (BBCH 73) 10days after pathogen inoculation (10 dai). Proteomic analysis using a two-dimensional differential gel electrophoresis (2D-DIGE) technique coupled with LC-MS/MS investigated 98 protein spots revealing quantitative or qualitative differences between the experimental variants. Protein functional annotation of 93 identified protein spots revealed that most affected functional groups represent storage proteins (globulins, hordeins), followed by proteins involved in carbohydrate metabolism (α-amylase inhibitor, β-amylase, glycolytic enzymes), amino acid metabolism (aminotransferases), defence response (chitinase, xylanase inhibitor, serpins, SGT1, universal stress protein USP), protein folding (chaperones, chaperonins), redox metabolism (ascorbate-glutathione cycle), and proteasome-dependent protein degradation. The obtained results indicate adverse effects of infection on plant proteome as well as an active plant response to pathogen as shown by enhanced levels of several inhibitors of pathogen-produced degradation enzymes (α-amylase inhibitor, xylanase inhibitor, serpins), chaperones, and other stress-related proteins (SGT1, USP). Genotypic differences were found in hordein abundance between Chevron and Pedant.

Discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2) is a novel biomarker of myxofibrosarcoma invasion identified by global protein expression profiling

Myxofibrosarcoma (MFS) is a mesenchymal malignancy characterized by frequent recurrence even after radical wide resection. To optimize therapy for MFS patients, we aimed to identify candidate tissue biomarkers of MFS invasion potential. Invasion characteristics of MFS were evaluated by magnetic resonance imaging and protein expression profiling of primary tumor tissues performed using two-dimensional difference gel electrophoresis (2D-DIGE). Protein expression profiles were compared between invasive and non-invasive tumors surgically resected from 11 patients. Among the 3453 protein spots observed, 59 demonstrated statistically significant difference in intensity (≥2-fold) between invasive and non-invasive tumors (p<0.01 by Wilkoxon test), and were identified by mass spectrometry as 47 individual proteins. Among them, we further focused on discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2), a receptor tyrosine kinase with aberrant expression in malignant tumors. Immunohistochemistry analysis of 21 additional MFS cases revealed that higher DCBLD2 expression was significantly associated with invasive properties of tumor cells. DCBLD2 sensitivity and specificity, and positive and negative predictive values for MFS invasion were 69.2%, 87.5%, 90%, and 63.6%, respectively. The expression level of DCBLD2 was consistent in different portions of tumor tissues. Thus, DCBLD2 expression can be a useful biomarker to evaluate invasive properties of MFS. Further validation studies based on multi-institutional collaboration and comprehensive analysis of DCBLD2 biological functions in MFS are required to confirm its prognostic utility for clinical application.

Melatonin ameliorates anxiety and depression-like behaviors and modulates proteomic changes in triple transgenic mice of Alzheimer's disease.

Alzheimer's disease (AD) is a devastating neurodegenerative disease accompanied by neuropsychiatric symptoms, such as anxiety and depression. The levels of melatonin decrease in brains of AD patients. The potential effect of melatonin on anxiety and depression behaviors in AD and the underlying mechanisms remain unclear. In this study, we treated 10-month-old triple transgenic mice of AD (3xTg-AD) with melatonin (10 mg/kg body weight/day) for 1 month and explored the effects of melatonin on anxiety and depression-like behaviors in 3xTg-AD mice and the protein expression of hippocampal tissues. The behavioral test showed that melatonin ameliorated anxiety and depression-like behaviors of 3xTg-AD mice as measured by open field test, elevated plus maze test, forced swimming test, and tail suspension test. By carrying out two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry, we revealed a total of 46 differentially expressed proteins in hippocampus between the wild-type (WT) mice and non-treated 3xTg-AD mice. A total of 21 differentially expressed proteins were revealed in hippocampus between melatonin-treated and non-treated 3xTg-AD mice. Among these differentially expressed proteins, glutathione S-transferase P 1 (GSTP1) (an anxiety-associated protein) and complexin-1 (CPLX1) (a depression-associated protein) were significantly down-regulated in hippocampus of 3xTg-AD mice compared with the WT mice. The expression of these two proteins was modulated by melatonin treatment. Our study suggested that melatonin could be used as a potential candidate drug to improve the neuropsychiatric behaviors in AD via modulating the expression of the proteins (i.e. GSTP1 and CPLX1) involved in anxiety and depression behaviors. © 2017 BioFactors, 43(4):593-611, 2017.

Proteomics of the epicardial fat secretome and its role in post-operative atrial fibrillation.

Post-operative atrial fibrillation (POAF) is a predictor of morbidity and mortality after cardiac surgery. Latent predisposing factors may reside in the epicardial adipose tissue (EAT) due to its anatomical position and high protein production rate. In order to explore a possible mechanistic link, we characterized proteins secreted by the EAT preceding the onset of POAF. Epicardial adipose tissue samples were collected from 76 consecutive patients with no history of AF undergoing coronary artery bypass surgery, 50 samples for proteomic analysis and 26 for gene expression studies, further divided according to development of POAF. Ten vs. 10 matched samples representing EAT secretome were analysed by two-dimensional difference in-gel electrophoresis (2D-DIGE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to identify differentially expressed proteins (P < 0.05, expression change >1.2 fold). Findings were validated by Western blotting on EAT protein extracts and by gene expression studies via quantitative polymerase chain reaction (qPCR). Proteomics returned 35 differentially expressed proteins. Amongst those, gelsolin was down regulated in POAF. Western blot analysis confirmed a significant reduction in gelsolin in the AF group. Gene expression for gelsolin was significantly reduced in the AF group confirming the proteomics findings. For the first time we describe EAT secretome as a possible substrate for POAF. It contains various proteins differentially expressed in patients who later develop POAF. Amongst those gelsolin, involved in inflammation and ion channel regulation, was associated with maintenance of sinus rhythm. Understanding the role of EAT may offer novel insights into prevention and treatment of AF.

Proteomic features linked to tenderness of aged pork loins.

There is considerable evidence that the protein component of fresh pork makes a major contribution to tenderness. In particular, the proteomic profile can be linked to postmortem events including pH decline, tissue oxidation, and protein degradation. The objectives for this study were to determine differences in sarcoplasmic proteomes that contribute to tenderness variation in aged pork longissimus dorsi muscles (LM). A defined set of pork loins selected to be similar in pH, color, and lipid yet different in tenderness were used. Pork loins were assigned to tenderness groups based on their star probe values; a high star probe group (HSP; n=12 mean star probe 7.75 kg) and low star probe group (LPS; n=12 star probe 4.95 kg) Samples were selected for proteomic experiments based on star probe values, and selected samples were within specified ranges for ultimate pH (5.54-5.86), marbling score (1.0-3.0), and percent total lipid (1.61-3.37%). Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry were used to examine sarcoplasmic protein abundance and potential modifications. Proteins spots that were significantly different across groups were selected for identification. Results from 2D-DIGE showed that HSP samples had significantly more abundant metabolic, stress response, and regulatory proteins in the sarcoplasmic fraction compared with LSP samples. The stress response protein peroxiredoxin-2 was more abundant in HSP samples as determined by 2D-DIGE ( ≤ 0.01; 2 spots) and western blot assay ( = 0.02). Low star probe samples showed significantly more degradation of the structural protein desmin in 2D-DIGE ( < 0.01) and western blot assay ( < 0.01). These results demonstrate that extreme proteolytic differences influenced measured tenderness of LSP and HSP samples and that soluble desmin and peroxiredoxin-2 may be used as biomarkers to differentiate between tough and tender aged pork products.

Proteomic profiling of peripheral blood neutrophils identifies two inflammatory phenotypes in stable COPD patients

BackgroundCOPD is a heterogeneous chronic inflammatory disease of the airways and it is well accepted that the GOLD classification does not fully represent the complex clinical manifestations of COPD and this classification therefore is not well suited for phenotyping of individual patients with COPD. Besides the chronic inflammation in the lung compartment, there is also a systemic inflammation present in COPD patients. This systemic inflammation is associated with elevated levels of cytokines in the peripheral blood, but the precise composition is unknown. Therefore, differences in phenotype of peripheral blood neutrophils in vivo could be used as a read out for the overall systemic inflammation in COPD.MethodOur aim was to utilize an unsupervised method to assess the proteomic profile of peripheral neutrophils of stable COPD patients and healthy age matched controls to find potential differences in these profiles as read-out of inflammatory phenotypes. We performed fluorescence two-dimensional difference gel electrophoresis with the lysates of peripheral neutrophils of controls and stable COPD patients.ResultsWe identified two groups of COPD patients based on the differentially regulated proteins and hierarchical clustering whereas there was no difference in lung function between these two COPD groups. The neutrophils from one of the COPD groups were less responsive to bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF).ConclusionThis illustrates that systemic inflammatory signals do not necessarily correlate with the GOLD classification and that inflammatory phenotyping can significantly add in an improved diagnosis of single COPD patients.Trial registrationClinicaltrials.gov: NCT00807469 registered December 11th 2008

Identification of potential biomarkers for systemic lupus erythematosus diagnosis using two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry

Systemic lupus erythematosus (SLE) is an autoimmune disease of the connective tissue with a large spectrum of clinical manifestations. Immune deregulation leads to autoantibody and immune complexes overproduction, complement activation, and persistent tissue inflammation. Considering that the current diagnosis depends on the interpretation of the complex criteria established by the American College of Rheumatology and that the disease course is characterized by unpredictable activations and remissions, each patient develops different manifestations, and therefore, the discovery of specific biomarkers is urgently required. Therefore, this study aimed to identify putative biomarkers for active and inactive SLE potentially capable in distinguishing laboratorial SLE from other autoimmune diseases. The 2D-DIGE proteomics technique was used to evaluate the differential abundance of proteins between patients with active SLE, inactive SLE, patients with other autoimmune disease, and healthy individuals. Six proteins showed increased abundance in active SLE (A) and inactive SLE (I) compared to the C and O groups, but not between groups A and I. There were two transthyretin (TTR) fragments or proteins with a structure similar to TTR (accession numbers: PDB: 1GKO_A and 2PAB_A), retinol-binding protein 4 (RBP4) isoform X1 (no information in databases such as UNIPROT), and antibody fragments. Two proteins, APO-AIV and SP-40,40, were upregulated in group A than in O and C and in group I versus C, but not in group I versus O. Therefore, we suggest these proteins to be considered as candidates for the diagnosis of SLE.