Effects of Fe deficiency on the protein profile of Brassica napus phloem sap

The aim of this work was to study the effect of Fe deficiency on the protein profile of phloem sap exudates from Brassica napus using 2DE (IEF‐SDS‐PAGE). The experiment was repeated thrice and two technical replicates per treatment were done. Phloem sap purity was assessed by measuring sugar concentrations. Two hundred sixty‐three spots were consistently detected and 15.6% (41) of them showed significant changes in relative abundance (22 decreasing and 19 increasing) as a result of Fe deficiency. Among them, 85% (35 spots), were unambiguously identified. Functional categories containing the largest number of protein species showing changes as a consequence of Fe deficiency were signaling and regulation (32%), and stress and redox homeostasis (17%). The Phloem sap showed a higher oxidative stress and significant changes in the hormonal profile as a result of Fe deficiency. Results indicate that Fe deficiency elicits major changes in signaling pathways involving Ca and hormones, which are generally associated with flowering and developmental processes, causes an alteration in ROS homeostasis processes, and induces decreases in the abundances of proteins involved in sieve element repair, suggesting that Fe‐deficient plants may have an impaired capacity to heal sieve elements upon injury.     

The secreted salivary proteome of the pea aphid Acyrthosiphon pisum characterised by mass spectrometry

Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE‐LC‐MS/MS and LC‐MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin‐converting enzyme (an M2 metalloprotease), an M1 zinc‐dependant metalloprotease, a glucose‐methanol‐choline (GMC)‐oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium‐binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium‐mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.     

Identification of differentially expressed proteins by treatment with PUGNAc in 3T3‐L1 adipocytes through analysis of ATP‐binding proteome

O‐GlcNAc (2‐acetamino‐2‐deoxy‐β‐D‐glucopyranose), an important modification for cellular processes, is catalyzed by O‐GlcNAc transferase and O‐GlcNAcase. O‐(2‐acetamido‐2‐deoxy‐D‐glucopyranosylidene) amino‐N‐phenylcarbamate (PUGNAc) is a nonselective inhibitor of O‐GlcNAcase, which increases the level of protein O‐GlcNAcylation and is known to induce insulin‐resistance in adipose cells due to uncharacterized targets of this inhibitor. In this study, using ATP affinity chromatography, we applied a targeted proteomic approach for identification of proteins induced by treatment with PUGNAc. For optimization of proteomic methods using ATP affinity chromatography, comparison of two cell lines (3T3‐L1 adipocytes and C2C12 myotubes) and two different digestion steps was performed using four different structures of immobilized ATP‐bound resins. Using this approach, based on DNA sequence homologies, we found that the identified proteins covered almost half of ATP‐binding protein families classified by PROSITE. The optimized ATP affinity chromatography approach was applied for identification of proteins that were differentially expressed in 3T3‐L1 adipocytes following treatment with PUGNAc. For label‐free quantitation, a gel‐assisted method was used for digestion of the eluted proteins, and analysis was performed using two different MS modes, data‐independent (671 proteins identified) and data‐dependent (533 proteins identified) analyses. Among identified proteins, 261 proteins belong to nucleotide‐binding proteins and we focused on some nucleotide‐binding proteins, ubiquitin‐activation enzyme 1 (E1), Hsp70, vasolin‐containing protein (Vcp), and Hsp90, involved in ubiquitin‐proteasome degradation and insulin signaling pathways. In addition, we found that treatment with PUGNAc resulted in increased ubiquitination of proteins in a time‐dependent manner, and a decrease in both the amount of Akt and the level of phosphorylation of Akt, a key component in insulin signaling, through downregulation of Hsp90. In this study, based on a targeted proteomic approach using ATP affinity chromatography, we found four proteins related to ubiquitination and insulin signaling pathways that were induced by treatment with PUGNAc. This result would provide insight into understanding functions of PUGNAc in 3T3‐L1 cells.     

Helicobacter pylori proteomics by 2‐DE/MS, 1‐DE‐LC/MS and functional data mining

With its predicted proteome of 1550 proteins (data set Etalon) Helicobacter pylori 26695 represents a perfect model system of medium complexity for investigating basic questions in proteomics. We analyzed urea‐solubilized proteins by 2‐DE/MS (data set 2‐DE) and by 1‐DE‐LC/MS (Supprot); proteins insoluble in 9 M urea but solubilized by SDS (Pellet); proteins precipitating in the Sephadex layer at the application side of IEF (Sephadex) by 1‐DE‐LC/MS; and proteins precipitating close to the application side within the IEF gel by LC/MS (Startline). The experimental proteomics data of H. pylori comprising 567 proteins (protein coverage: 36.6%) were stored in the Proteome Database System for Microbial Research ( http://www.mpiib‐berlin.mpg.de/2D‐PAGE/ ), which gives access to raw mass spectra (MALDI‐TOF/TOF) in T2D format, as well as to text files of peak lists. For data mining the protein mapping and comparison tool PROMPT ( http://webclu.bio.wzw.tum.de/prompt/ ) was used. The percentage of proteins with transmembrane regions, relative to all proteins detected, was 0, 0.2, 0, 0.5, 3.8 and 6.3% for 2‐DE, Supprot, Startline, Sephadex, Pellet, and Etalon, respectively. 2‐DE does not separate membrane proteins because they are insoluble in 9 M urea/70 mM DTT and 2% CHAPS. SDS solubilizes a considerable portion of the urea‐insoluble proteins and makes them accessible for separation by SDS‐PAGE and LC. The 2‐DE/MS analysis with urea‐solubilized proteins and the 1‐DE‐LC/MS analysis with the urea‐insoluble protein fraction (Pellet) are complementary procedures in the pursuit of a complete proteome analysis. Access to the PROMPT‐generated diagrams in the Proteome Database allows the mining of experimental data with respect to other functional aspects.     

Comprehensive proteome profiling of the Fe(III)‐reducing myxobacterium Anaeromyxobacter dehalogenans 2CP‐C during growth with fumarate and ferric citrate

Anaeromyxobacter dehalogenans is a microaerophilic member of the delta‐proteobacteria which is able to utilize a wide range of electron acceptors, including halogenated phenols, U(VI), Fe(III), nitrate, nitrite, oxygen and fumarate. To date, the knowledge regarding general metabolic activities of this ecologically relevant bacterium is limited. Here, we present a first systematic 2‐D reference map of the soluble A. dehalogenans proteome in order to provide a sound basis for further proteomic studies as well as to gain first global insights into the metabolic activities of this bacterium. Using a combination of 2‐DE and MALDI‐TOF‐MS, a total of 720 proteins spots were identified, representing 559 unique protein species. Using the proteome data, altogether 50 metabolic pathways were found to be expressed during growth with fumarate as primary electron acceptor. An analysis of the pathways revealed an extensive display of enzymes involved in the catabolism and anabolism of a variety of amino acids, including the unexpected fermentation of lysine to butyrate. Moreover, using the reference gel as basis, a semi‐quantitative analysis of protein expression changes of A. dehalogenans during growth with ferric citrate as electron acceptor was conducted. The adaptation to Fe(III) reducing conditions involved the expression changes of a total of 239 proteins. The results suggest that the adaptation to Fe(III) reductive conditions involves an increase in metabolic flux through the tricarboxylic acid cycle, which is fueled by an increased catabolism of amino acids.     

Pumpkin eIF5A isoforms interact with components of the translational machinery in the cucurbit sieve tube system

In yeast, eIF5A, in combination with eEF2, functions at the translation step, during the protein elongation cycle. This result is of significance with respect to functioning of the enucleate sieve tube system, as eIF5A was recently detected in Cucurbita maxima (pumpkin) phloem sap. In the present study, we further characterized four CmeIF5A isoforms, encoding three proteins, all of which were present in the phloem sap. Although hypusination of CmeIF5A was not necessary for entry into the sieve elements, this unique post‐translational modification was necessary for RNA binding. The two enzymes required for hypusination were detected in pumpkin phloem sap, where presumably this modification takes place. A combination of gel‐filtration chromatography and protein overlay assays demonstrated that, as in yeast, CmeIF5A interacts with phloem proteins, like eEF2, known to be involved in protein synthesis. These findings are discussed in terms of a potential role for eIF5A in regulating protein synthesis within the enucleate sieve tube system of the angiosperms.     

Proteomic profiling of N‐linked glycoproteins identifies ConA‐binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N‐linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty‐eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high‐levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA‐binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, “ConA‐binding proCD” (ConA‐pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5–5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA‐pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConA‐pCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.     

Protein extraction from the earthworm Eisenia fetida for 2‐DE

We identified an efficient protocol for extracting proteins from whole earthworm, Eisenia fetida, for 2‐DE. Sample preparation is a critical step in a 2‐DE proteome approach and is absolutely essential for obtaining good results. Six protein extraction protocols based on different protein precipitation agents were tested and evaluated using 2‐DE. The methods generated remarkably different 2‐DE protein spot patterns. We conclude that trichloroacetic acid (TCA)‐A eliminates interfering compounds, thus allowing for the efficient resolubilization of proteins. TCA‐A gives good distinction, more bands in 1‐DE gels, and the most number of protein spots in 2‐DE gels. It is also rapid, provides the higher protein yield, and has the less number of steps. To demonstrate the quality of the extracted proteins, we cut several protein spots that were common to four methods from 2‐DE gels, analyzed them using MALDI‐TOF/TOF MS, and tentatively identified them. The classic TCA‐A method proved to be most useful as a standard method of extracting proteins from E. fetida.     

Identification of Zn-nicotianamine and Fe-2'-Deoxymugineic acid in the phloem sap from rice plants (Oryza sativa L.)

In higher plants, the supply of metals such as Zn and Fe via phloem is important for the growth and physiology of young organs. However, little information is available on the speciation (chemical forms) of these metals in the phloem fluids. Because the pH of phloem fluids is slightly alkaline and the concentration of phosphate, which may bind to metals, is high, Zn and Fe in phloem fluids could be precipitated if these metals do not form complexes with some ligand compounds. In the present experiment, we examined the chemical forms of Zn and Fe in phloem sap collected from rice (Oryza sativa L.) by separating the phloem sap using size-exclusion and anion-exchange chromatography, and identifying the contents using electrospray ionization time-of-flight mass spectrometry. The low molecular weight chemical forms of Zn and Fe were identified as Zn-nicotianamine and Fe(III)-2'-deoxymugineic acid complexes, respectively. This report is the first to identify metal-chelate complexes in rice phloem sap.     

Identification of inositol 1,4,5‐trisphosphate‐binding proteins by heparin‐agarose affinity purification and LTQ ORBITRAP MS in Oryza sativa

Inositol 1,4,5‐trisphosohate (IP₃) and its receptors play a pivotal role in calcium signal transduction in mammals. However, no homologs of mammalian IP₃ receptors have been found in plants. In this study, we isolated the microsomal fractions from rice cells in suspension culture and further obtained putative IP₃‐binding proteins by heparin‐agarose affinity purification. The IP₃‐binding activities of these protein fractions were determined by [³H] IP₃‐binding assay. SDS‐PAGE and MS analysis were then performed to characterize these proteins. We have identified 297 proteins from the eluates of heparin‐agarose column chromatography, which will provide insight into the IP₃ signaling pathways in plants. All MS data have been deposited in the ProteomeXchange with identifier PXD000763 ( http://proteomecentral.proteomexchange.org/dataset/PXD000763 ).     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Faba bean forisomes can function in defence against generalist aphids

Phloem sieve elements have shut‐off mechanisms that prevent loss of nutrient‐rich phloem sap when the phloem is damaged. Some phloem proteins such as the proteins that form forisomes in legume sieve elements are one such mechanism and in response to damage, they instantly form occlusions that stop the flow of sap. It has long been hypothesized that one function of phloem proteins is defence against phloem sap‐feeding insects such as aphids. This study provides the first experimental evidence that aphid feeding can induce phloem protein occlusion and that the aphid‐induced occlusions inhibit phloem sap ingestion. The great majority of phloem penetrations in Vicia faba by the generalist aphids Myzus persicae and Macrosiphum euphorbiae triggered forisome occlusion and the aphids eventually withdrew their stylets without ingesting phloem sap. This contrasts starkly with a previous study on the legume‐specialist aphid, Acyrthosiphon pisum, where penetration of faba bean sieve elements did not trigger forisome occlusion and the aphids readily ingested phloem sap. Next, forisome occlusion was demonstrated to be the cause of failed phloem ingestion attempts by M. persicae: when occlusion was inhibited by the calcium channel blocker lanthanum, M. persicae readily ingested faba bean phloem sap.     

A comparative study on the heparin‐binding proteomes of Toxoplasma gondii and Plasmodium falciparum

Toxoplasma gondii is an obligatory intracellular apicomplexan parasite which exploits host cell surface components in cell invasion and intracellular parasitization. Sulfated glycans such as heparin and heparan sulfate have been reported to inhibit cell invasion by T. gondii and other apicomplexan parasites such as Plasmodium falciparum. The aim of this study was to investigate the heparin‐binding proteome of T. gondii. The parasite‐derived components were affinity‐purified on the heparin moiety followed by MS fingerprinting of the proteins. The heparin‐binding proteins of T. gondii and P. falciparum were compared based on functionality and affinity to heparin. Among the proteins identified, the invasion‐related parasite ligands derived from tachyzoite/merozoite surface and the secretory organelles were prominent. However, the profiles of the proteins were different in terms of affinity to heparin. In T. gondii, the proteins with highest affinity to heparin were the intracellular components with functions of parasite development contrasted to that of P. falciparum, of which the rhoptry‐derived proteins were prominently identified. The profiling of the heparin‐binding proteins of the two apicomplexan parasites not only explained the mechanism of heparin‐mediated host cell invasion inhibition, but also, to a certain extent, revealed that the action of heparin on the parasite extended after endocytosis.     

Pre‐fractionation of rat liver cytosol proteins prior to mass spectrometry‐based proteomic analysis using tandem biomimetic affinity chromatography

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low‐copy‐number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non‐bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre‐fractionation were digested and then analyzed by LC‐MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un‐fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un‐fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre‐fractionation with MS‐based proteomic analysis is simple, low‐cost, and effective, providing the prospect of broad application in proteomics. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two‐dimensional gel electrophoresis

Introduction – A variety of sample preparation protocols for plant proteomic analysis using two‐dimensional gel electrophoresis (2‐DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. Objective – This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2‐DE. Methodology – Four sample preparation methods were tested: (1) phenol extraction and methanol–ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid–acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS–PAGE (1‐DE) and 2‐DE. Fifteen selected protein spots were trypsinised and analysed by matrix‐assisted laser desorption/ionisation time‐of‐flight tandem mass spectrometry (MALDI‐TOF‐MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Results – Methods number 3 and 4 resulted in large quantities of protein with good 1‐DE separation and were chosen for 2‐DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. Conclusion – The described sample preparation method allows the proteomic analysis of papaya leaves by 2‐DE and mass spectrometry (MALDI‐TOF‐MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species. Copyright © 2009 John Wiley & Sons, Ltd.     

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.