Comparative Proteomic Approach Research Articles

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker.

A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey-bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease-chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments.

Identification of CALM as the Potential Serum Biomarker for Predicting Recurrence of Nasopharyngeal Carcinoma Using Mass Spectrometry-Based Comparative Proteomic Approach

To date, there are no serum biomarkers for prediction of recurrent nasopharyngeal carcinoma (rNPC). Diagnosis of rNPC depends on imaging and biopsy of diseased tissue mostly, both two ways work mostly if target tumor in an advanced stage. Therefore, predicting recurrent biomarkers are urgently required. In this study, we used tandem mass tags(TMT) labeling and high performance liquid chromatography(HPLC)fractionation followed by liquid chromatograph-tandem mass spectrometry (LC-MS/MS) analysis to identify differentially expressed proteins, and serum was collected from 40 nasopharyngeal carcinoma (NPC)patients (recurrence (n = 20) and no-recurrence (n = 20). Compared to nrNPC, we found 59 proteins significantly dysregulated in rNPC, most were previously reported to play a role in carcinogenesis. Dysregulation of Calmodulinis (CALM) was confirmed in 74 new patients (recurrence (n = 32) and no-recurrence (n = 42) by ELISA. Moreover, we performed a preliminary pathway analysis showed Oxidative phosphorylation was altered in rNPC compared to nrNPC patients. These data together identify potential diagnostic biomarker for rNPC and elucidate the potential molecular mechanisms that are dysregulated and contribute to the pathogenesis of rNPC.

Drought and salinity induced changes in ecophysiology and proteomic profile of Parthenium hysterophorus.

Parthenium hysterophorus is a plant that tolerates drought and salinity to an extremely high degree. Higher expression of stress-responsive proteome contributes for greater defence against abiotic stresses. Thus, P. hysterophorus could be a rich source of genes that encode stress-imparting mechanisms and systems. The present study utilizes comparative physiological and proteomic approaches for identification of key proteins involved in stress-defence of P. hysterophorus. Thirty-days-old plants were exposed to drought (10% PEG 6000) and salinity (160 mM NaCl) for 10 days duration. Both stresses induced oxidative stress estimated in terms of TBARS and H2O2. Levels of both enzymatic and non-enzymatic antioxidants were elevated, more by drought than salinity. Particularly, SOD, GR, CAT and GST proved to be assisting as very commendable defence under drought, as well as salinity. Levels of ascorbate, glutathione and proline were also increased by both stresses, more in response to drought. Comparative proteomics analysis revealed a significant change in relative abundance of 72 proteins under drought and salinity. Drought and salinity increased abundance of 45 and 41 proteins and decreased abundance of 24 and 26 proteins, respectively. Drought and salinity increased and decreased abundance of 31 and 18 proteins, respectively. The functions of identified proteins included those related to defence response (26%), signal transduction (13%), transcription and translation (10%), growth and development (8.5%), photosynthesis (8.5%), metabolism (7%), terpenoid biosynthesis (5.5%), protein modification and transport (7%), oxido-reductase (4%) and Miscellaneous (11%). Among the defence related proteins, antioxidants and HSPs constituted 26% and 21%, respectively. Present study suggests a potential role of defence proteins. Proteins involved in molecular stabilization, formation of osmolytes and wax and contributing to stress-avoiding anatomical features emerged as key and complex mechanisms for imparting stress tolerance to P. hysterophorus.

Comparative proteomics of cancer stem cells in osteosarcoma using ultra-high-performance liquid chromatography and Orbitrap Fusion mass spectrometer

Osteosarcoma is the most common malignant tumour found in bones, and it has a poor prognosis. For improved therapy, it is significant to have a deep understanding of the proteomics changes in the cancer stem cells (CSCs) of osteosarcoma. Therefore, a comparative proteomics approach based on ultra-high-performance liquid chromatography coupled to an Orbitrap Fusion mass spectrometer (UHPLC-Orbitrap Fusion MS) was established to investigate the key molecular changes between CSCs and non-CSCs in human osteosarcoma HOS cells. A proteomic analysis was performed on these samples and a total of more than 6600 proteins were identified in each run. Moreover, most of the correlation coefficients between three biological repeats were more than 0.9 in each group. That finding demonstrated not only that the reproducibility of the test is good but also that the stability of this MS is excellent. A label-free quantitative method was applied to analyse differentially expressed proteins. Using the criteria of greater than 1.5-fold changes and a p value < 0.05, 124 proteins were identified as being significantly different between HOS-CSCs and non-CSCs. A pathway analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed the potential molecular regulatory networks that may regulate CSCs. Selected differential α-actinin 4 (ACTN4) proteins were validated by Western blot assay. These findings enhance our understanding of the molecular changes in CSCs and may provide additional improvements in therapy for treating osteosarcoma. Moreover, the UHPLC-Orbitrap Fusion MS-based proteomics method is helpful in cancer research.

Differences between Platelets Derived from Neonatal Cord Blood and Adult Peripheral Blood Assessed by Mass Spectrometry.

It has been proposed that differences may exist between umbilical cord blood (CB) platelets and adult peripheral blood (APB) platelets, including altered protein levels of the main platelet integrins. We have now compared the protein expression profiles of CB and APB platelets employing a label-free comparative proteomics approach. Aggregation studies showed that CB platelets effectively aggregate in the presence of thromboxane A2 analogue, collagen, and peptide agonists of the proteinase-activated receptors 1 and 4. In agreement with previous studies, higher concentrations of the agonists were required to initiate aggregation in the CB platelets. Mass spectrometry analysis revealed no significant difference in the expression levels of critical platelet receptors like glycoprotein (GP)Ib, GPV, GPIX, and integrin αIIbβ3. This was confirmed using flow cytometry-based approaches. Gene ontology enrichment analysis revealed that elevated proteins in CB platelets were in particular enriched in proteins contributing to mitochondrial energy metabolism processes. The reduced proteins were enriched in proteins involved in, among others, platelet degranulation and activation. In conclusion, this study reveals that the CB and APB platelets are distinct. In particular, changes were observed for proteins that belong to metabolic and energy generation processes and not for the critical adhesive platelet integrins and glycoproteins.

Persulfidation proteome reveals the regulation of protein function by hydrogen sulfide in diverse biological processes in Arabidopsis.

Hydrogen sulfide-mediated signaling pathways regulate many physiological and pathophysiological processes in mammalian and plant systems. The molecular mechanism by which hydrogen sulfide exerts its action involves the post-translational modification of cysteine residues to form a persulfidated thiol motif, a process called protein persulfidation. We have developed a comparative and quantitative proteomic analysis approach for the detection of endogenous persulfidated proteins in wild-type Arabidopsis and L-CYSTEINE DESULFHYDRASE 1 mutant leaves using the tag-switch method. The 2015 identified persulfidated proteins were isolated from plants grown under controlled conditions, and therefore, at least 5% of the entire Arabidopsis proteome may undergo persulfidation under baseline conditions. Bioinformatic analysis revealed that persulfidated cysteines participate in a wide range of biological functions, regulating important processes such as carbon metabolism, plant responses to abiotic and biotic stresses, plant growth and development, and RNA translation. Quantitative analysis in both genetic backgrounds reveals that protein persulfidation is mainly involved in primary metabolic pathways such as the tricarboxylic acid cycle, glycolysis, and the Calvin cycle, suggesting that this protein modification is a new regulatory component in these pathways.

The significance of proline and glutamate on butanol chaotropic stress in Bacillus subtilis 168

BackgroundButanol is an intensively used industrial solvent and an attractive alternative biofuel, but the bioproduction suffers from its high toxicity. Among the native butanol producers and heterologous butanol-producing hosts, Bacillus subtilis 168 exhibited relatively higher butanol tolerance. Nevertheless, organic solvent tolerance mechanisms in Bacilli and Gram-positive bacteria have relatively less information. Thus, this study aimed to elucidate butanol stress responses that may involve in unique tolerance of B. subtilis 168 to butanol and other alcohol biocommodities.ResultsUsing comparative proteomics approach and molecular analysis of butanol-challenged B. subtilis 168, 108 butanol-responsive proteins were revealed, and classified into seven groups according to their biological functions. While parts of them may be similar to the proteins reportedly involved in solvent stress response in other Gram-positive bacteria, significant role of proline in the proline–glutamate–arginine metabolism was substantiated. Detection of intracellular proline and glutamate accumulation, as well as glutamate transient conversion during butanol exposure confirmed their necessity, especially proline, for cellular butanol tolerance. Disruption of the particular genes in proline biosynthesis pathways clarified the essential role of the anabolic ProB-ProA-ProI system over the osmoadaptive ProH-ProA-ProJ system for cellular protection in response to butanol exposure. Molecular modifications to increase gene dosage for proline biosynthesis as well as for glutamate acquisition enhanced butanol tolerance of B. subtilis 168 up to 1.8% (vol/vol) under the conditions tested.ConclusionThis work revealed the important role of proline as an effective compatible solute that is required to protect cells against butanol chaotropic effect and to maintain cellular functions in B. subtilis 168 during butanol exposure. Nevertheless, the accumulation of intracellular proline against butanol stress required a metabolic conversion of glutamate through the specific biosynthetic ProB-ProA-ProI route. Thus, exogenous addition of glutamate, but not proline, enhanced butanol tolerance. These findings serve as a practical knowledge to enhance B. subtilis 168 butanol tolerance, and demonstrate means to engineer the bacterial host to promote higher butanol/alcohol tolerance of B. subtilis 168 for the production of butanol and other alcohol biocommodities.

Gel-free/label-free proteomic, photosynthetic, and biochemical analysis of cowpea (Vigna unguiculata [L.] Walp.) resistance against Cowpea severe mosaic virus (CPSMV)

Cowpea severe mosaic virus (CPSMV) causes significant losses in cowpea (Vigna unguiculata) production. In this present study biochemical, physiological, and proteomic analysis were done to identify pathways and defense proteins that are altered during the incompatible interaction between the cowpea genotype BRS-Marataoã and CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V) were evaluated at 2 and 6days post-inoculation (DPI). Data support the assumptions that increases in biochemical (high hydrogen peroxide, antioxidant enzymes, and secondary compounds) and physiological responses (high photosynthesis index and chlorophyll content), confirmed by label-free comparative proteomic approach, in which quantitative changes in proteasome proteins, proteins related to photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were observed may be implicated in the resistance of BRS-Marataoã to CPSMV. This pioneering study provides information for the selection of specific pathways and proteins, altered in this incompatible relationship, which could be chosen as targets for detailed studies to advance our understanding of the molecular, physiological, and biochemistry basis of the resistance mechanism of cowpea and design approachs to engineer plants that are more productive. Biological significanceThis is a pioneering study in which an incompatible relationship between a resistant cowpea and Cowpea severe mosaic virus (CPSMV) was conducted to comparatively evaluate proteomic profiles by Gel-free/label-free methodology and some physiological and biochemical parameters to shed light on how a resistant cowpea cultivar deals with the virus attack. Specific proteins and associated pathways were altered in the cowpea plants challenged with CPSMV and will contribute to our knowledge on the biological process tailored by cowpea in response to CPSMV.

Proteomic Analysis of Compatible Interaction between Sorghum Downy MildewPathogen (Peronosclerospora Sorghi) and Maize (Zea MaysL.)

Proteomic Analysis of Compatible Interaction between Sorghum Downy Mildew Pathogen (Peronosclerospora Sorghi) and Maize (Zea Mays L.) Sorghum downy mildew (SDM) caused by the oomycete, Peronosclerospora sorghi, is one of the destructive diseases that afflict maize. P. sorghi infects a susceptible host, hindering its growth and altering its morphology. To understand the molecular basis of the compatibility interaction between P. sorghi and maize, a comparative proteomic approach (2D-PAGE) was employed between the mock-inoculated (control) and SDM-inoculated leaves in the susceptible genotypes of maize (UMI79 and CM500). Seventeen spots showed a significant difference in the abundance of proteins in control and inoculated samples were further analyzed with MALDITOF/MS. The resulting peptide mass fingerprint was subjected to MASCOT analysis and it was found that most were related to stress that includes lipoxygenase and DEAD-box ATP-dependent RNA helicases, microtubule-associated protein and probable protein disulphide isomerase. Additionally, proteins involved in the cell cycle/endoreplication such as DNA topoisomerase 6 subunit A and retinoblastoma-related proteins were differentially expressed during infection. The possible roles of these proteins in response to P. sorghi infection in maize are discussed...

Comparative Proteomics Reveals Differences in Host-Pathogen Interaction between Infectious and Commensal Relationship with Campylobacter jejuni.

Campylobacter jejuni is the leading food-borne poisoning in industrialized countries. While the bacteria causes disease in humans, it merely colonizes the gut in poultry or pigs, where seems to establish a commensal relationship. Until now, few studies have been conducted to elucidate the relationship between C. jejuni and its different hosts. In this work, a comparative proteomics approach was used to identify the underlying mechanisms involved in the divergent outcome following C. jejuni infection in human and porcine host. Human (INT-407) and porcine (IPEC-1) intestinal cell lines were infected by C. jejuni for 3 h (T3h) and 24 h (T24h). C. jejuni infection prompted an intense inflammatory response at T3h in human intestinal cells, mainly characterized by expression of proteins involved in cell spreading, cell migration and promotion of reactive oxygen species (ROS). Proteomic analysis evidenced significantly regulated biofunctions in human cells related with engulfment and endocytosis, and supported by canonical pathways associated to infection such as caveolar- and clathrin-mediated endocytosis signaling. In porcine IPEC-1 cells, inflammatory response as well as signaling pathways that control cellular functions such as cell migration, endocytosis and cell cycle progression resulted downregulated. These differences in the host response to infection were supported by the different pattern of adhesion and invasion proteins expressed by C. jejuni in human and porcine cells. No marked differences in expression of virulence factors involved in adaptive response and iron acquisition functions were observed. Therefore, the results of this study suggest that both host and pathogen factors are responsible for commensal or infectious character of C. jejuni in different hosts.

Proteomic Analyses of Changes in Synechococcus sp. PCC7942 Following UV-C Stress.

UV-C's effects on the physiological and biochemical processes of cyanobacteria have been well characterized. However, the molecular mechanisms of cyanobacteria's tolerance to UV-C still need further investigation. This research attempts to decode the variation in protein abundances in cyanobacteria after UV-C stress. Different expression levels of proteins in the cytoplasm of Synechococcus sp. PCC7942 under UV-C stress were investigated using a comparative proteomic approach. In total, 47 UV-C-regulated proteins were identified by MALDI-TOF analysis and classified by Gene Ontology (GO). After studying their pathways, the proteins were mainly enriched in the groups of protein folding, inorganic ion transport and energy production. By focusing on these areas, this study reveals the correlation between UV-C stress-responsive proteins and the physiological changes of Synechococcus sp. PCC7942 under UV-C radiation. These findings may open up new areas for further exploration in the homeostatic mechanisms associated with cyanobacteria responses to UV-C radiation.

Aquatic adaptation of a laterally acquired pectin degradation pathway in marine gammaproteobacteria.

Mobile genomic islands distribute functional traits between microbes and habitats, yet it remains unclear how their proteins adapt to new environments. Here we used a comparative phylogenomic and proteomic approach to show that the marine bacterium Pseudoalteromonas haloplanktis ANT/505 acquired a genomic island with a functional pathway for pectin catabolism. Bioinformatics and biochemical experiments revealed that this pathway encodes a series of carbohydrate-active enzymes including two multi-modular pectate lyases, PelA and PelB. PelA is a large enzyme with a polysaccharide lyase family 1 (PL1)domain and a carbohydrate esterase family 8 domain, and PelB contains a PL1 domain and two carbohydrate-binding domains of family 13. Comparative phylogenomic analyses indicate that the pathway was most likely acquired from terrestrial microbes, yet we observed multi-modular orthologues only in marine bacteria. Proteomic experiments showed that P. haloplanktis ANT/505 secretes both pectate lyases into the environment in the presence of pectin. These multi-modular enzymes may therefore represent a marine innovation that enhances physical interaction with pectins to reduce loss of substrate and enzymes by diffusion. Our results revealed that marine bacteria can catabolize pectin, and highlight enzyme fusion as a potential adaptation that may facilitate microbial consumption of polymeric substrates in aquatic environments.

Identification of proteins with different abundance associated with cell migration and proliferation in leiomyoma interstitial fluid by proteomics.

Uterine leiomyoma is the most common female reproductive tract benign tumor. Little is known about protein composition and changes in the leiomyoma interstitial fluid (IF). The present study focused on changes in protein abundance in the IF of leiomyoma. Leiomyoma IFs and adjacent myometrial IFs were obtained and analyzed by two-dimensional electrophoresis (2-DE) coupled with mass spectrometry and western blotting for 2-DE data validation. A total of 25 unique proteins were observed to change significantly (P<0.05). Of these proteins with different abundance, 22 had not been previously identified in leiomyoma IF. In silico analysis predicted that three of these proteins were secreted via classical mechanisms, while 22 were secreted via non-classical mechanisms. Ingenuity Pathway Analysis identified 17 proteins associated with cellular migration and proliferation. Among these, phosphoglycerate mutase 1 had not been previously associated with leiomyoma. The abundance of seven proteins was further validated by western blotting. A comparative proteomic approach identified a number of proteins associated with cellular migration and proliferation, with changes in abundance in IF likely to be involved in tumor development. Further studies will be required to investigate the role of these proteins in leiomyoma IF and their possible association with tumor development and growth.

Proteomics analysis reveals the molecular mechanism underlying the transition from primary to secondary growth of poplar

Wood is the most important natural source of energy and also provides fuel and fiber. Considering the significant role of wood, it is critical to understand how wood is formed. Integration of knowledge about wood development at the cellular and molecular levels will allow more comprehensive understanding of this complex process. In the present study, we used a comparative proteomic approach to investigate the differences in protein profiles between primary and secondary growth in young poplar stems using tandem mass tag (TMT)-labeling. More than 10,816 proteins were identified, and, among these, 3106 proteins were differentially expressed during primary to secondary growth. Proteomic data were validated using a combination of histochemical staining, enzyme activity assays, and quantitative real-time PCR. Bioinformatics analysis revealed that these differentially expressed proteins are related to various metabolic pathways, mainly including signaling, phytohormones, cell cycle, cell wall, secondary metabolism, carbohydrate and energy metabolism, and protein metabolism as well as redox and stress pathways. This large proteomics dataset will be valuable for uncovering the molecular changes occurring during the transition from primary to secondary growth. Further, it provides new and accurate information for tree breeding to modify wood properties.

Chemical Proteomics Maps Brain Region Specific Activity of Endocannabinoid Hydrolases.

The biosynthetic and catabolic enzymes of the endocannabinoids tightly regulate endocannabinoid-mediated activation of the cannabinoid CB1 receptor. Monitoring the activities of these endocannabinoid hydrolases in different brain regions is, therefore, key to gaining insight into spatiotemporal control of CB1 receptor-mediated physiology. We have employed a comparative chemical proteomics approach to quantitatively map the activity profile of endocannabinoid hydrolases in various mouse brain regions at the same time. To this end, we used two different activity-based probes: fluorophosphonate-biotin (FP-biotin), which quantifies FAAH, ABHD6, and MAG-lipase activity, and MB108, which detects DAGL-α, ABHD4, ABHD6, and ABHD12. In total, 32 serine hydrolases were evaluated in the frontal cortex, hippocampus, striatum, and cerebellum. Comparison of endocannabinoid hydrolase activity in the four brain regions revealed that FAAH activity was highest in the hippocampus, and MAGL activity was most pronounced in the frontal cortex, whereas DAGL-α was most active in the cerebellum. Comparison of the activity profiles with a global proteomics data set revealed pronounced differences. This could indicate that post-translational modification of the endocannabinoid hydrolases is important to regulate their activity. Next, the effect of genetic deletion of the CB1 receptor was studied. No difference in the enzymatic activity was found in the cerebellum, striatum, frontal cortex, and hippocampus of CB1 receptor knockout animals compared to wild type mice. Our results are in line with previous reports and indicate that the CB1 receptor exerts no regulatory control over the basal production and degradation of endocannabinoids and that genetic deletion of the CB1 receptor does not induce compensatory mechanisms in endocannabinoid hydrolase activity.

Identification of differentially expressed proteins in retinoblastoma tumors using mass spectrometry-based comparative proteomic approach

In India, retinoblastoma is among the top five childhood cancers. Children mostly present with extraocular extension and high risk features that results in unsatisfactory treatment and low survival rate. In addition, lack of potential therapeutic and prognostic targets is another challenge in the management of retinoblastoma. We studied comparative proteome of retinoblastoma patients (HPV positive and negative (n=4 each) and controls (n=4), in order to identify potential retinoblastoma-specific protein targets. 2D-DIGE coupled MALDI-TOF/TOF mass spectrometry identified 39 unique proteins. Highly deregulated proteins were GFAP,RBP3,APOA1,CRYAA,CRABP1,SAG and TF. Gene ontology (Panther 7.0) revealed majority of proteins to be associated with metabolic processes (26%) and catalytic activity (38%). 8 proteins were significantly upregulated in HPV positive vis-a-vis HPV negative cases. Patient group exhibited 12 upregulated and 18 downregulated proteins compared to controls. Pathway and network analysis (IPA software) revealed CTNNB1 as most significantly regulated signalling pathway in HPV positive than HPV negative retinoblastoma. The trends in transcriptional change of 9 genes were consistent with those at proteomic level. The Western blot analysis confirmed the expression pattern of RBP3,GFAP and CRABP1. We suggest GFAP,RBP3,CRABP1,CRYAAA,APOA1 and SAG as prospective targets that could further be explored as potential candidates in therapy and may further assist in studying the disease mechanism. SignificanceIn this study we evaluated tumor tissue specimens from retinoblastoma patients and identified 39 differentially regulated proteins compared to healthy retina. From these, we propose RBP3, CRABP1, GFAP, CRYAA, APOA1 and SAG as promising proteomic signatures that could further be explored as efficient prognostic and therapeutic targets in retinoblastoma. The present study is not only a contribution to the ongoing endeavour for the discovery of proteomic signatures in retinoblastoma, but, may also act as a starting point for future studies aimed at uncovering novel targets for further therapeutic interventions and improving patient outcomes.

Tick-host conflict: immunoglobulin E antibodies to tick proteins in patients with anaphylaxis to tick bite.

Tick-borne infectious diseases and allergies are a growing problem worldwide. Tick bite allergy has been associated with the direct effect of immunoglobulin E (IgE) response to tick salivary antigens, or secondary to the induction of allergy to red meat consumption through IgE antibodies against the carbohydrate α-Gal (Gal α 1-3Gal β 1-(3)4GlcNAc-R). However, despite the growing burden of this pathology, the proteins associated with anaphylaxis to tick bite have not been characterized. To address this question, a comparative proteomics approach was used to characterize tick proteins producing an IgE antibody response in a healthy individual with record of tick bites, which had not resulted in any allergic reactions, and two patients with anaphylactic reactions to Rhipicephalus bursa or Hyalomma marginatum tick bites. Both patients and the healthy individual were red meat tolerant. The results supported a patient-specific IgE antibody response to tick species responsible for the anaphylaxis to tick bite. Both patients and the healthy individual serologically recognized tick proteins with and without α-Gal modifications, with proteins differentially recognized by patients but not control sera. These proteins could be used as potential antigens for diagnostics, treatment and prevention of tick bite-induced allergies.

Comparative iTRAQ proteomic profiling of susceptible and resistant apple cultivars infected by Alternaria alternata apple pathotype

Alternaria blotch, caused by the Alternaria alternata apple pathotype (A. alternata AP), is one of serious pathogen of apples. In order to better understand the molecular mechanisms that underlie the defense responses of apple resistance to Alternaria blotch disease, a comparative proteomic approach was applied to analyze of susceptible and resistant apple cultivars response to A. alternata AP infection using iTRAQ (isobaric tags for relative and absolute quantitation) technique. A total of 4225 proteins were identified, and 1226 proteins were quantified. Of the quantified proteins, 280 and 34 expressed differentially (fold change >1.5) at 72 h post-infection (HPI) in the susceptible (“Starking Delicious”) and the resistant (“Jonathan”) apple cultivars, respectively, compared with mock-inoculated controls. Most of the differentially expressed proteins (DEPs) were associated with host plant resistance to pathogens, including signal transduction, stress and defense, and photosynthesis metabolism. Among these proteins, beta-1,3-glucanase(PR2), thaumatin-like protein (PR5), and lipoxygenase were found in both susceptible and resistant hosts. However, endochitinase and (+)-neomenthol dehydrogenase were only detected in the resistant cultivar and increased in abundance in response to the pathogen attack. To study the role of pathogenesis-related (PR) proteins in the early infection process, their expressions at 6, 18, 36, and 72 HPI were analyzed by western blot. It showed that PR5 were accumulated to a high level at 6 HPI in “Jonathan,” while cannot be detected in “Starking Delicious” until 18 HPI. The above results suggested that endochitinase and (+)-neomenthol dehydrogenase, as well as PR5 which exerts function at early stage, play important roles in apple plant against A. alternata AP infestation.

Comparative proteomic analysis of leaf tissue from tomato plants colonized with Rhizophagus irregularis

A comparative proteomic approach was performed to analyze the differential accumulation of leaf proteins in response to the symbiosis between Solanum lycopersicum and the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis. Protein profiling was examined in leaves from tomato plants colonized with AMF (M), as well as non-colonized plants fertilized with low phosphate (20 μM P; NM-LP) and non-colonized plants fertilized with regular phosphate Hoagland’s solution (200 μM P; NM-RP). Comparisons were made between these groups, and 2D-SDS-PAGE revealed that 27 spots were differentially accumulated in M vs. NM-LP. Twenty-three out of the 27 spots were successfully identified by mass spectrometry. Two of these proteins, 2-methylene-furan-3-one reductase and auxin-binding protein ABP19a, were up-accumulated in M plants. The down-accumulated proteins in M plants were associated mainly with photosynthesis, redox, and other molecular functions. Superoxide dismutase, harpin binding protein, and thioredoxin peroxidase were down-accumulated in leaves of M tomato plants when compared to NM-LP and NM-RP, indicating that these proteins are responsive to AMF colonization independently of the phosphate regime under which they were grown. 14-3-3 protein was up-accumulated in NM-RP vs. NM-LP plants, whereas it was down-accumulated in M vs. NM-LP and M vs. NM-RP, regardless of their phosphate nutrition. This suggests a possible regulation by P nutrition and AMF colonization. Our results demonstrate AMF-induced systemic changes in the expression of tomato leaf proteins, including the down-accumulation of proteins related to photosynthesis and redox function.

Biocompatibility of iron carbide and detection of metals ions signaling proteomic analysis via HPLC/ESI-Orbitrap

Recently, magnetic nanoparticles (NPs) have been extensively used in food industry and biomedical treatments. However, the biocompatibility mechanism on expression proteomics, before consideration of magnetic NPs for clinical application, has not yet been fully elucidated. Therefore, this study was undertaken to identify potential biomarkers of metal ion signaling proteins in human cervical cancer cell line (HeLa) cells. Here, we report the in vitro investigations of the cell cycle response and significant changes in protein abundance of HeLa cells when exposed to self-tailored hydrophilic Fe2C NPs. The comparative proteomic approach based on 18O labeling coupled with high performance liquid chromatography/electrospray ionization with ion trap mass analyzer (HPLC/ESI-Orbitrap) was applied, and 394 proteins were identified. There were 46 significantly differentiated proteins based on the specific metal ion signaling response. Among them, 60S ribosomal protein L37a, serine/arginine-rich splicing factor 7, calmodulin, and calumenin were downregulated, whereas transketolase was overexpressed. Functional interaction network of Fe2C-regulated proteins was successfully created by the STRING algorithm to show the strong interactions between proteins. This work will not only help to understand the molecular mechanism of metal ion signaling proteins that can potentially be used to develop therapeutic protocols for diagnosis of diseases but also give direction for tailoring biocompatible magnetic NPs.