Identification of kaempferol‐regulated proteins in rat calvarial osteoblasts during mineralization by proteomics

Kaempferol, a flavonoid, promotes osteoblast mineralization in vitro and bone formation in vivo; however, its mechanism of action is yet unknown. We adopted proteomic approach to identify the differential effect of kaempferol on rat primary calvarial osteoblasts during mineralization. The primary rat calvarial osteoblasts were treated with kaempferol (5.0 μM) for 9 days under mineralizing condition that resulted in significant increase in alkaline phosphatase activity and mineralization of the cells. Further, 2‐D analysis of the kaempferol‐treated osteoblast lysates revealed 18 differentially expressed proteins (nine upregulated and nine downregulated) on the basis of >/<2.0‐fold as cut‐off (p<0.01) that were then identified by MALDI‐TOF MS. These included cytoskeletal proteins, intracellular signaling protein, chaperone, extracellular matrix protein, and proteins involved in glycolysis and cell–matrix interactions. Proteomics data were confirmed by Western blotting and quantitative real‐time PCR by randomly selecting two upregulated and two downregulated proteins. Western blot analysis confirmed upregulation of HSP‐70 and cytokeratin‐14 levels, and downregulation of aldose reductase and caldesmon expression. We further demonstrated that kaempferol treatment inhibits aldose reductase activity in osteoblasts indicating an altered cellular metabolism by decelerating polyol pathway that was associated with the kaempferol‐induced osteoblast mineralization. In conclusion, this is a first comprehensive study on the differential regulation of proteins by kaempferol in primary osteoblast, which would further help to elucidate the role of the identified proteins in the process of osteoblast mineralization.     

Quantitative Analysis of the Global Proteome in Peripheral Blood Mononuclear Cells from Patients with New‐Onset Psoriasis

Psoriasis is a common chronic autoimmune skin disease involving the activation of T cells. To explore the proteomic signature of peripheral blood mononuclear cells, a quantitative analysis of their global proteome was conducted in samples from Chinese patients with new‐onset psoriasis (n = 31) and healthy controls (n = 32) using an integrated quantitative approach with tandem mass tag labeling and LC–MS/MS. Protein annotation, unsupervised hierarchical clustering, functional classification, functional enrichment and cluster, and protein–protein interaction analyses were performed. A total of 5178 proteins were identified, of which 4404 proteins were quantified. The fold‐change cutoff was set at 1.2 (patients vs controls); 335 proteins were upregulated, and 107 proteins were downregulated. The bioinformatics analysis indicated that the differentially expressed proteins were involved in processes related to the activation of immune cells including the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) pathway, cellular energy metabolism, and proliferation. Three upregulated proteins and two phosphorylated proteins in the NF‐κB pathway were verified or identified by Western blotting. These results confirm that the NF‐κB pathway is critical to psoriasis. In addition, many differentially expressed proteins identified in this study have never before been associated with psoriasis, and further studies on these proteins are necessary.     

Proteomic analysis of the sea-island cotton roots infected by wilt pathogen Verticillium dahliae

Verticillium wilt of cotton is a vascular disease mainly caused by the soil-born filamentous fungus Verticillium dahliae. To study the mechanisms associated with defense responses in wilt-resistant sea-island cotton (Gossypium barbadense) upon V. dahliae infection, a comparative proteomic analysis between infected and mock-inoculated roots of G. barbadense var. Hai 7124 (a cultivar showing resistance against V. dahliae) was performed by 2-DE combined with local EST database-assisted PMF and MS/MS analysis. A total of 51 upregulated and 17 downregulated proteins were identified, and these proteins are mainly involved in defense and stress responses, primary and secondary metabolisms, lipid transport, and cytoskeleton organization. Three novel clues regarding wilt resistance of G. barbadense are gained from this study. First, ethylene signaling was significantly activated in the cotton roots attacked by V. dahliae as shown by the elevated expression of ethylene biosynthesis and signaling components. Second, the Bet v 1 family proteins may play an important role in the defense reaction against Verticillium wilt. Third, wilt resistance may implicate the redirection of carbohydrate flux from glycolysis to pentose phosphate pathway (PPP). To our knowledge, this study is the first root proteomic analysis on cotton wilt resistance and provides important insights for establishing strategies to control this disease.     

Plasma proteomic analysis of patients infected with H1N1 influenza virus

This study profiled the plasma proteins of patients infected by the 2011 H1N1 influenza virus. Differential protein expression was identified in plasma obtained from noninfected control subjects (n = 15) and H1N1‐infected subjects (n = 15). Plasma proteins were separated by a 2DE large gel system and identified by nano‐ultra performance LC‐MS. Western blot assays were performed to validate proteins. Eight plasma proteins were upregulated and six proteins were downregulated among 3316 plasma proteins in the H1N1‐infected group as compared with the control group. Of 14 up‐ and downregulated proteins, nine plasma proteins were validated by Western blot analysis. Putative protein FAM 157A, leucine‐rich alpha 2 glycoprotein, serum amyloid A protein, and dual oxidase 1 showed significant differential expression. The identified plasma proteins could be potential candidates for biomarkers of H1N1 influenza viral infection. Further studies are needed to develop these proteins as diagnostic biomarkers.     

Proteomic analysis of Medicago truncatula root plastids

Despite the recognized importance of non‐photosynthetic plastids in a wide array of plant processes, the root plastid proteome of soil‐grown plants still remains to be explored. In this study, we used a protocol allowing the isolation of Medicago truncatula root plastids with sufficient protein recovery and purity for their subsequent in‐depth analysis by nanoscale capillary LC‐MS/MS. Besides providing the first picture of a root plastid proteome, the results obtained highlighted the identification of 266 protein candidates whose functional distribution mainly resembled that of wheat endosperm amyloplasts and tobacco proplastids together with displaying major differences to those reported for chloroplasts. Most of the identified proteins have a role in nucleic acid‐related processes (16%), carbohydrate (15%) and nitrogen/sulphur (12%) metabolisms together with stress response mechanisms (10%). It is noteworthy that BLAST searches performed against the proteins reported in different plastidomes allowed detecting 30 putative root plastid proteins for which homologues were previously unsuspected as plastid‐located, most of them displaying a common putative role in participating in the plant cell responses against abiotic and/or biotic stresses. Taken together, the data obtained provide new insights into the functioning of root plastids and reinforce the emerging idea for an important role of these organelles in sustaining plant defence reactions.     

Comparative analyses of three legume species reveals conserved and unique root extracellular proteins

Increasing evidence suggests that root extracellular proteins are involved in interactions between roots and their soil environment. In the present study, exudates released by 6‐day‐old roots of the three legume species white lupin (Lupinus albus), soybean (Glycine max), and cowpea (Vigna sinensis) were collected under axenic conditions, and their constitutively secreted proteomes were analyzed. Between 42 and 93 unique root extracellular proteins with 2 or more different peptide fragments per protein were identified by LC‐MS/MS. Functional annotation of these proteins classified them into 14–16 different functional categories. Among those 14 homologous proteins were identified in at least two legume species. Among the unique proteins, 58 in white lupin, 85 in soybean, and 31 in cowpea were specific for each plant species, and many of them were classified in the same functional categories. Interestingly, in contrast to soybean and cowpea, two protein bands of approximately 16 and 30 kDa were present on the SDS‐PAGE gel of white lupin. The identification of these bands revealed a class III chitinase and a thaumatin‐like protein. Both belong to the class of pathogenesis‐related proteins. The results imply that root extracellular proteins play important roles in the cross‐talk between plant roots and the rhizosphere.     

The association of a distinct plasma proteomic profile with the cervical high-grade squamous intraepithelial lesion of Uyghur women: a 2D liquid-phase chromatography/mass spectrometry study

Objective: To identify plasma protein biomarkers of cervical high-grade squamous intraepithelial lesion (HSIL) of Uyghur women by proteomics approach.

Methods: Plasma protein samples of Uyghur women with HSIL and chronic cervicitis were analyzed with 2D HPLC followed by detection of target proteins with Linear Trap Quadrupole Mass Spectrometer (LTQ MS/MS).

Results: We detected three upregulated and one downregulated protein peaks representing protein constituents distinguishing HSIL from controls by 2D HPLC, identified 31 target proteins by LTQ MS/MS. Further confirmed analysis with online software IPA^® 8.7 and ELISA assay showed APOA1 and mTOR as potential biomarkers.

Conclusions: A distinct plasma proteomic profile may be associated with HSIL of Uyghur women.     

Comparative Proteomic Profiling of Methicillin‐Susceptible and Resistant Staphylococcus aureus

Staphylococcus aureus is a highly successful human pathogen responsible for a wide range of infections. This study provides insights into the virulence, pathogenicity, and antimicrobial resistance determinants of methicillin‐susceptible and methicillin‐resistant S. aureus (MSSA; MRSA) recovered from non‐healthcare environments. Three environmental MSSA and three environmental MRSA are selected for proteomic profiling using isobaric tag for relative and absolute quantitation tandem mass spectrometry (iTRAQ MS/MS). Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway annotation are applied to interpret the functions of the proteins detected. 792 proteins are identified in MSSA and MRSA. Comparative analysis of MRSA and MSSA reveals that 8 of out 792 proteins are upregulated and 156 are downregulated. Proteins that have differences in abundance are predominantly involved in catalytic and binding activity. Among 164 differently abundant proteins, 29 are involved in pathogenesis, antimicrobial resistance, stress response, mismatch repair, and cell wall synthesis. Twenty‐two proteins associated with pathogenicity including SPA, SBI, CLFA, and DLT are upregulated in MRSA. Moreover, the upregulated pathogenic protein ENTC2 in MSSA is determined to be a super antigen, potentially capable of triggering toxic shock syndrome in the host. Enhanced pathogenicity, antimicrobial resistance, and stress response are observed in MRSA compared to MSSA.     

A secreted chitinase‐like protein (OsCLP) supports root growth through calcium signaling in Oryza sativa

Chitinases belong to a conserved protein family and play multiple roles in defense, development and growth regulation in plants. Here, we identified a secreted chitinase‐like protein, OsCLP, which functions in rice growth. A T‐DNA insertion mutant of OsCLP (osclp) showed significant retardation of root and shoot growth. A comparative proteomic analysis was carried out using root tissue of wild‐type and the osclp mutant to understand the OsCLP‐mediated rice growth retardation. Results obtained revealed that proteins related to glycolysis (phosphoglycerate kinase), stress adaption (chaperonin) and calcium signaling (calreticulin and CDPK1) were differentially regulated in osclp roots. Fura‐2 molecular probe staining, which is an intracellular calcium indicator, and inductively coupled plasma‐mass spectrometry (ICP‐MS) analysis suggested that the intracellular calcium content was significantly lower in roots of osclp as compared with the wild‐type. Exogenous application of Ca²⁺ resulted in successful recovery of both primary and lateral root growth in osclp. Moreover, overexpression of OsCLP resulted in improved growth with modified seed shape and starch structure; however, the overall yield remained unaffected. Taken together, our results highlight the involvement of OsCLP in rice growth by regulating the intracellular calcium concentrations.     

Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ‐coupled two‐dimensional LC‐MS/MS

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis is a chronic disease. Currently, there are no sufficiently validated biomarkers for early diagnosis of TB infection. In this study, a panel of potential serum biomarkers was identified between patients with pulmonary TB and healthy controls by using iTRAQ‐coupled 2D LC‐MS/MS technique. Among 100 differentially expressed proteins screened, 45 proteins were upregulated (>1.25‐fold at p < 0.05) and 55 proteins were downregulated (<0.8‐fold at p < 0.05) in the TB serum. Bioinformatics analysis revealed that the differentially expressed proteins were related to the response to stimulus, the metabolic and immune system processes. The significantly differential expression of apolipoprotein CII (APOCII), CD5 antigen‐like (CD5L), hyaluronan‐binding protein 2 (HABP2), and retinol‐binding protein 4 (RBP4) was further confirmed using immunoblotting and ELISA analysis. By forward stepwise multivariate regression analysis, a panel of serum biomarkers including APOCII, CD5L, and RBP4 was obtained to form the disease diagnostic model. The receiver operation characteristic curve of the diagnostic model was 0.98 (sensitivity = 93.42%, specificity = 92.86%). In conclusion, APOCII, CD5L, HABP2, and RBP4 may be potential protein biomarkers of pulmonary TB. Our research provides useful data for early diagnosis of TB.     

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel‐based and a LC MS/MS‐based proteomics method. Two‐day‐old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two‐phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel‐based proteomics, four and eight protein spots were identified as up‐ and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up‐ and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low‐abundance proteins could be identified by the LC MS/MS‐based method. Three homologues of plasma membrane H⁺‐ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H⁺‐ATPase protein.     

Nep1‐like protein from Moniliophthora perniciosa induces a rapid proteome and metabolome reprogramming in cells of Nicotiana benthamiana

NEP1 (necrosis‐ and ethylene‐inducing peptide 1)‐like proteins (NLPs) have been identified in a variety of taxonomically unrelated plant pathogens and share a common characteristic of inducing responses of plant defense and cell death in dicotyledonous plants. Even though some aspects of NLP action have been well characterized, nothing is known about the global range of modifications in proteome and metabolome of NLP‐treated plant cells. Here, using both proteomic and metabolomic approaches we were able to identify the global molecular and biochemical changes in cells of Nicotiana benthamiana elicited by short‐term treatment with MpNEP2, a NLP of Moniliophthora perniciosa, the basidiomycete responsible for the witches' broom disease on cocoa (Theobroma cacao L.). Approximately 100 protein spots were collected from 2‐DE gels in each proteome, with one‐third showing more than twofold differences in the expression values. Fifty‐three such proteins were identified by mass spectrometry (MS)/MS and mapped into specific metabolic pathways and cellular processes. Most MpNEP2 upregulated proteins are involved in nucleotide‐binding function and oxidoreductase activity, whereas the downregulated proteins are mostly involved in glycolysis, response to stress and protein folding. Thirty metabolites were detected by gas spectrometry (GC)/MS and semi‐quantified, of which eleven showed significant differences between the treatments, including proline, alanine, myo‐inositol, ethylene, threonine and hydroxylamine. The global changes described affect the reduction‐oxidation reactions, ATP biosynthesis and key signaling molecules as calcium and hydrogen peroxide. These findings will help creating a broader understanding of NLP‐mediated cell death signaling in plants.     

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi‐lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2‐DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2‐DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole‐time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin‐related proteins, F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF‐induced morphological change of MSCs.     

Proteomics analysis of liver samples from puffer fish Takifugu rubripes exposed to excessive fluoride: An insight into molecular response to fluorosis

Comparative proteomics was performed to identify proteins in the liver of Takifugu rubripes in response to excessive fluoride exposure. Sixteen fish were randomly divided into a control group and an experimental group. The control group was raised in soft water alone (F^? = 0.4 mg/L), and the experimental group was raised in the same water with sodium fluoride at a high concentration of 35 mg/L. After 3 days, proteins were extracted from the fish livers and then subjected to two‐dimensional polyacrylamide gel electrophoresis analysis. The matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was applied to identify the proteins that were differentially expressed from the two groups of fish. Among an average of 816 and 918 proteins detected in the control and treated groups, respectively, 16 proteins were upregulated and 35 were downregulated (P < 0.01) in the fluoride‐treated group as compared with those in the control group. Twenty‐four highly differentially expressed proteins were further analyzed by MALDI‐TOF/TOF‐MS, and eight were identified by Mascot. These eight proteins include disulfide isomerase ER‐60, 4SNc‐Tudor domain protein, SMC3 protein, Cyclin D1, and mitogen‐activated protein kinase 10, as well as three unknown proteins. Consistent with their previously known functions, these identified proteins seem to be involved in apoptosis and other functions associated with fluorosis. These results will greatly contribute to our understanding of the effects of fluoride exposure on the physiological and biochemical functions of Takifugu and the toxicological mechanism of fluoride causing fluorosis in both fish and human. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:21–28, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20308     

Proteomic analysis of human papillomavirus‐related oral squamous cell carcinoma: Identification of thioredoxin and epidermal‐fatty acid binding protein as upregulated protein markers in microdissected tumor tissue

Human papillomavirus (HPV) infection has been identified as an etiologic agent for a subset of oral squamous cell carcinoma (OSCC) with increasing incidence. HPV DNA‐positivity may confer better prognosis but the related oncogenic mechanisms are unknown. For the identification of HPV relevant proteins, we analyzed microdissected cells from HPV DNA‐positive (n = 17) and HPV DNA‐negative (n = 7) OSCC tissue samples. We identified 18 proteins from tumor tissues by peptide fingerprint mapping and SELDI MS that were separated using 2‐DE. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified thioredoxin (TRX) and epidermal‐fatty acid binding protein as upregulated in HPV related tumor tissue. This study, investigating for the first time proteomic changes in microdissected HPV infected tumor tissue, provides an indication on the oncogenic potential of viruses.     

Proteome profiling of aging in mouse models: Differential expression of proteins involved in metabolism,transport, and stress response in kidney

Aging is a time‐dependent complex biological phenomenon observed in various organs and organelles of all living organisms. To understand the molecular mechanism of age‐associated functional loss in aging kidneys, we have analyzed the expression of proteins in the kidneys of young (19–22 wk) and old (24 months) C57/BL6 male mice using 2‐DE followed by LC‐MS/MS. We found that expression levels of 49 proteins were upregulated (p ≤ 0.05), while that of only ten proteins were downregulated (p ≤ 0.05) due to aging. The proteins identified belong to three broad functional categories: (i) metabolism (e.g., aldehyde dehydrogenase family, ATP synthase β‐subunit, malate dehydrogenase, NADH dehydrogenase (ubiquinone), hydroxy acid oxidase 2), (ii) transport (e.g., transferrin), and (iii) chaperone/stress response (e.g., Ig‐binding protein, low density lipoprotein receptor‐related protein associated protein 1, selenium‐binding proteins (SBPs)). Some proteins with unknown functions were also identified as being differentially expressed. ATP synthase β subunit, transferrin, fumarate hydratase, SBPs, and albumin are present in multiple forms, possibly arising due to proteolysis or PTMs. The above functional categories suggest specific mechanisms and pathways for age‐related kidney degeneration.