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.     

Noninvasive biomarkers of endometriosis: myth or reality?

Endometriosis affects 10% of premenopausal women and 35–50% of women with infertility, pelvic pain, or both. At present, endometriosis can only be diagnosed with surgery, where laparoscopy is considered a gold standard. Noninvasive biomarkers are thus urgently needed. In 2010, the peripheral biomarkers of endometriosis were systematically reviewed by May et al. However, with the introduction of ‘-omics’ technologies, we have witnessed immense progress in biomarker discovery, which now calls for an overview of recent studies. This report looks at potential blood and urine biomarkers of endometriosis published in the last 3 years. The current status of noninvasive diagnostic biomarkers of endometriosis is discussed, with the limitations of these studies identified and recommendations for future biomarker discovery provided.     

Proteomic Serum Profile of Fatigued Men Receiving Localized External Beam Radiation Therapy for Non-Metastatic Prostate Cancer

ContextFatigue is the most distressing side effect of radiation therapy, and its progression etiology is unknown.ObjectivesThis study describes proteome changes from sera of fatigued men with non-metastatic prostate cancer receiving external beam radiation therapy (EBRT).MethodsFatigue scores, measured by the Functional Assessment of Chronic Illness Therapy-Fatigue, and serum were collected from 12 subjects at baseline (before EBRT) and at midpoint (Day 21) of EBRT. Depleted sera from both time points were analyzed using two-dimensional difference gel electrophoresis, and up/down regulated proteins were identified using liquid chromatography-tandem mass spectrometry. Western blot analyses confirmed the protein changes observed.ResultsResults showed that apolipoprotein (Apo)A1, ApoE, and transthyretin (TTR) consistently changed from baseline (Day 0) to midpoint (Day 21). The mean ApoE level of subjects with high change in fatigue (HF: n = 9) increased significantly from baseline to midpoint and were higher than in subjects with no change in fatigue. The mean ApoA1 level was higher in HF subjects at baseline and at midpoint than in no fatigue subjects at both time points. The mean TTR level of no fatigue subjects was higher at baseline and midpoint than in HF subjects.ConclusionThese ApoE, ApoA1, and TTR results may assist in understanding pathways that can explain fatigue progression etiology in this clinical population.     

Histology‐guided protein digestion/extraction from formalin‐fixed and paraffin‐embedded pressure ulcer biopsies

Herein we present a simple, reproducible and versatile approach for in situ protein digestion and identification on formalin‐fixed and paraffin‐embedded (FFPE) tissues. This adaptation is based on the use of an enzyme delivery platform (hydrogel discs) that can be positioned on the surface of a tissue section. By simultaneous deposition of multiple hydrogels over select regions of interest within the same tissue section, multiple peptide extracts can be obtained from discrete histological areas. After enzymatic digestion, the hydrogel extracts are submitted for LC‐MS/MS analysis followed by database inquiry for protein identification. Further, imaging mass spectrometry (IMS) is used to reveal the spatial distribution of the identified peptides within a serial tissue section. Optimization was achieved using cutaneous tissue from surgically excised pressure ulcers that were subdivided into two prime regions of interest: the wound bed and the adjacent dermal area. The robust display of tryptic peptides within these spectral analyses of histologically defined tissue regions suggests that LC‐MS/MS in combination with IMS can serve as useful exploratory tools.     

Protein profiles of cardiomyocyte differentiation in murine embryonic stem cells exposed to perfluorooctane sulfonate

Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may affect diverse systems in animals and humans, including the cardiovascular system. However, little is known about the mechanism by which it affects the biological systems. Herein, we used embryonic stem cell test procedure as a tool to assess the developmental cardiotoxicity of PFOS. The differentially expressed proteins were identified by quantitative proteomics that combines the stable isotope labeling of amino acids with high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry. Results of the embryonic stem cell test procedure suggested that PFOS was a weak embryotoxic chemical. Nevertheless, a few marker proteins related to cardiovascular development (Brachyury, GATA4, MEF2C, α‐actinin) were significantly reduced by exposure to PFOS. In total, 176 differential proteins were identified by proteomics analysis, of which 67 were upregulated and 109 were downregulated. Gene ontology annotation classified these proteins into 13 groups by molecular functions, 12 groups by cellular locations and 10 groups by biological processes. Most proteins were mainly relevant to either catalytic activity (25.6%), nucleus localization (28.9%) or to cellular component organization (19.8%). Pathway analysis revealed that 32 signaling pathways were affected, particularly these involved in metabolism. Changes in five proteins, including L ‐threonine dehydrogenase, X‐ray repair cross‐complementing 5, superoxide dismutase 2, and DNA methyltransferase 3b and 3a were confirmed by Western blotting, suggesting the reliability of the technique. These results revealed potential new targets of PFOS on the developmental cardiovascular system. Copyright © 2015 John Wiley & Sons, Ltd.     

Protein expression in the brain of rat offspring in relation to prenatal caloric restriction

Objective: Intrauterine growth restriction (IUGR) has been associated with decreased supply of crucial substrates to the fetus and affects its growth and development by temporarily or permanently modifying gene expression and function. However, not all neonates born by calorie restricted mothers are IUGR and there are no reports regarding their brain protein expression vis-à-vis that of their IUGR siblings. Here, we investigated the expression of key proteins that regulate growth and development of the brain in non-IUGR newborn pups versus IUGR siblings and control pups.

Methods: Rat brain proteins were isolated from each group upon delivery and separated by two-dimensional gel electrophoresis (2-DE).

Results: 14-3-3 Protein, calreticulin, elongation factor, alpha-enolase, fascin, heat-shock protein HSP90 and pyruvate kinase isozymes were significantly increased (p?<?0.05) in samples obtained from IUGR newborn pups compared to non-IUGR. Conversely, collapsin response mediator proteins, heat-shock70 and peroxiredoxin2 were decreased in IUGR group compared to non-IUGR.

Conclusions: In our experimental study, IUGR pups showed an altered proteomic profile compared to their non-IUGR siblings and non-IUGR controls. Thus, not all offspring of calorie-restricted mothers become IUGR with the accompanying alterations in the expression of proteins. The differentially expressed proteins could modulate alterations in the energy balance, plasticity and maturation of the brain.