MALDI-TOF/TOF Mass Spectrometry Research Articles

Identification of New Allergens in Macadamia Nut and Cross-Reactivity with Other Tree Nuts in a Spanish Cohort.

The consumption of macadamia nuts has increased due to their cardioprotective and antioxidant properties. However, this rise is consistent with an increase in the cases of macadamia nut allergy, leading to severe reactions. Although two Macadamia integrifolia allergens (Mac i 1 and Mac i 2) have been identified in Australian and Japanese patients, the allergenic sensitization patterns in Western European populations, particularly in Spain, remain unclear. For this purpose, seven patients with macadamia nut allergy were recruited in Spain. Macadamia nut protein extracts were prepared and, together with hazelnut and walnut extracts, were used in Western blot and inhibition assays. IgE-reactive proteins were identified using MALDI-TOF/TOF mass spectrometry (MS). Immunoblotting assays revealed various IgE-binding proteins in macadamia nut extracts. Mass spectrometry identified three new allergens: an oleosin, a pectin acetylesterase, and an aspartyl protease. Cross-reactivity studies showed that hazelnut extract but not walnut extract inhibited macadamia nut oleosin-specific IgE binding. This suggests that oleosin could be used as marker for macadamia-hazelnut cross-reactivity. The results show an allergenic profile in the Spanish cohort different from that previously detected in Australian and Japanese populations. The distinct sensitization profiles observed highlight the potential influence of dietary habits and environmental factors exposure on allergenicity.

Comparative proteomics analysis of the head proteins of worker honey bees (Apis mellifera) in production stage of royal jelly

Honey bees can be evaluated to study their economic, developmental and immunity aspects. Apis mellifera has important organs such as the hypopharyngeal gland, mandibular gland and brain in their heads. The hypopharyngeal glands and mandibular glands cooperate to produce royal jelly essential in feeding queen honey bees and broods. Moreover, the brain is an organ that regulates the social behaviors of honey bees such as memory and learning. Therefore, this study aimed to detect differential head protein changes in one-day-old and nine-day-old worker honey bees. 153 repeatable protein spots were identified using Melanie software and statistically analyzed. Of the total protein spots, 27 responsive spots were identified using MALDI TOF-TOF mass spectrometry, of which 14 and 8 spots showed up and down regulation, respectively and five spots were expressed just in the head capsules of nine-day-old workers. All identified proteins were divided into 16 different groups involved in various growth-related processes as well as royal jelly augmentation. Nine proteins including KMT5B, TGIF2LX, Protein mono-ADP-ribosyltransferase PARP12, TCL1B4, maph-9, Vimentin-type intermediate filament-associated coiled-coil protein, HSP68, tetratricopeptide repeat domain 36 and POC1B were also identified and introduced for the first time in A. mellifera. This study provides valuable information on proteins involved in honey bee head growth and development as well as increasing royal jelly production.

Proteomic changes of aryl hydrocarbon receptor (AhR)-silenced porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment and affecting human/animal health and reproduction. Intracellular TCDD action usually involves the activation of aryl hydrocarbon receptor (AhR). The aim of the current study was to examine TCDD-induced changes in the proteome of AhR-silenced porcine granulosa cells. The AhR-silenced cells were treated with TCDD (100 nM) for 3, 12 or 24 h. Total protein was isolated, labeled with cyanines and next, the samples were separated by isoelectric focusing and SDS-PAGE. Proteins of interest were identified by MALDI-TOF/TOF mass spectrometry (MS) analysis and confirmed by western blotting and fluorescence immunocytochemistry. The AhR-targeted siRNA transfection reduced the granulosal expression level of AhR by 60–70%. In AhR-silenced porcine granulosa cells, TCDD influenced the abundance of only three proteins: annexin V, protein disulfide isomerase and ATP synthase subunit beta. The obtained results revealed the ability of TCDD to alter protein abundance in an AhR-independent manner. This study offers a new insight into the mechanism of TCDD action and provide directions for future functional studies focused on molecular effects exerted by TCDD.

Capture and Analysis of Cell Surface N-Glycans by Hydrazide-Modified Magnetic Beads and CE-LIF

Cell surface proteins are important target molecules for biomarkers discovery. Diseases are often reflected within the glycan profiles of proteins. As such, studying the glycan profiles of cell surface proteins may give rise to new biomarker or drug target discoveries. Focused on cell surface glycoproteins, hydrazine technology had been applied to cell surface protein capturing (CSC) technology. In this study, hydrazide-functionalized magnetic beads were used to capture surface glycoproteins on intact cells and the released N-glycan profiling, except protein profiling, was investigated by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. The use of magnetic beads gave rapid separation and enrichment of the captured glycoproteins. The sample processing parameters, including the ratio of cells to hydrazine groups, oxidation time and cell incubation time with magnetic beads, cell lysis and glycopeptide cleavage methods, were evaluated using MALDI-TOF–TOF mass spectrometry analysis. Subsequently, citric acid was applied at optimized temperature for the glycan cleavage from the beads. The technique developed has the potential in sensing, diagnostic and therapeutic applications by speeding up the method development of glycoprotein identification, N-glycan profiling, drug target screening and biomarker discovery.

Abstract 2585: Immunoseroproteomic profiling in autoantibodies to ENO1 as potential biomarkers in immunodiagnosis of osteosarcoma by serological proteome analysis (SERPA) approach

Abstract Background: Osteosarcoma (OS) is the most common highly malignant primary solid bone-tumor. Despite its relatively low incidence rate among overall cancers, it remains one of the most harmful primary malignant tumors in childhood and adolescence. It is now evident that serum autoantibodies against tumor-associated antigens (TAAs) could be used as serological cancer biomarkers in types of cancers. Methods: Serological proteome analysis (SERPA) approach was applied to profile anti-TAA autoantibody responses in sera from patients with OS, and Normal Human, and explore differences of these responses. This approach can detect autoantibodies to TAAs that could serve as clinical biomarkers and immunotherapeutic agents. Sera from OS, Osteochondroma (OC) and Normal Human Sera (NHS) were probed by immunoblotting against cellular proteins extracted from U2-OS and Saos-2 cell lines, with OS sera showing stronger immunoreactivity. MALDI-TOF/TOF Mass Spectrometry (MS) analysis of immunoreactive protein spots revealed that several OS sera contained autoantibodies to a number of proteins, particularly to alpha-enolase (ENO1). Results: Analysis of 172 serum samples from patients with OS, OC and NHS by ELISA showed higher frequency of anti-ENO1 autoantibodies in OS sera compared to others. Interestingly, descant of ENO1 immunoreactivity was observed in most patients after treatments, which may imply a potential association between anti-ENO1 autoantibody titers and disease progression. Nine of twelve sera reacted strongly against purified ENO1, but 3 weakly against purified ENO1, which indicates 75.0% sera with positive optimal density values from ELISA were consistently positive in Western blotting. The expression of ENO1 in Osteosarcoma tissues was evaluated by immunohistochemistry in tumor microarray. Conclusion: Our intriguing findings firstly demonstrate that ENO1 is one of autoantigens that elicit autoimmune responses in OS and can be used as biomarkers in immunodiagnosis and progression of OS. Key words: Osteosarcoma (OS), Tumor-associated antigen (TAA), Serological proteome analysis (SERPA), ENO1, Immunodiagnosis. Citation Format: Jitian Li, Manyu Huang, Liping Dai, Zongchang Han, Xiaofei Qin, Wen Xie, Wei Qian, Wuyin Li, Jianying Zhang. Immunoseroproteomic profiling in autoantibodies to ENO1 as potential biomarkers in immunodiagnosis of osteosarcoma by serological proteome analysis (SERPA) approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2585.

Beta Actin Expression Profile in Malignant Human Glioma Tumors

Background: Proteomics is considered a new era in neurophysiological/ neuropathological research including brain tumors. Gliomas which are derived from glial cells are the most common type of brain tumor in humans. Methods: In the present study the total protein content of healthy cells of the brain and brain tumor cells was extracted, purified and quantified by Bradford assay. Two-dimensional electrophoresis were used for protein separation followed by statistical analysis. Primary protein detection was performed based on the differences in isoelectric pH, molecular weight of proteins and protein data banks, which was further confirmed by MALDI-TOF/TOF mass spectrometry (MS). Results: Our results showed elevated levels of beta-actin protein expression in glioma brain tumor cells. It is important to know when a cell is transformed and when it becomes malignant. Here we evaluated the beta-actin expression in malignant cells. Conclusion: Since structural changes are highly involved in tumor cell malignancy, beta-actin elevations can contribute in glioma tumor cell invasiveness.

Inhibition of Breast Cancer Cell Migration by Cyclotides Isolated from Pombalia calceolaria.

Two new bracelet cyclotides from roots of Pombalia calceolaria with potential anticancer activity have been characterized in this work. The cyclotides Poca A and B (1 and 2) and the previously known CyO4 (3) were de novo sequenced by MALDI-TOF/TOF mass spectrometry (MS). The MS2 spectra were examined and the amino acid sequences were determined. The purified peptides were tested for their cytotoxicity and effects on cell migration of MDA-MB-231, a triple-negative breast cancer cell line. The isolated cyclotides reduced the number of cancer cells by more than 80% at 20 μM, and the concentration-related cytotoxic responses were observed with IC50 values of 1.8, 2.7, and 9.8 μM for Poca A (1), Poca B (2), and CyO4 (3), respectively. Additionally, the inhibition of cell migration (wound-healing assay) exhibited that CyO4 (3) presents an interesting activity profile, in being able to inhibit cell migration (50%) at a subtoxic concentration (2 μM). The distribution of these cyclotides in the roots was analyzed by MALDI imaging, demonstrating that all three compounds are present in the phloem and cortical parenchyma regions.

Influence of different sample preparation strategies on the proteomic identification of stress biomarkers in porcine saliva

BackgroundThe influence of two different sample treatments comprising the enrichment of glycoproteins by boronic acid and dynamic range compression by hexapeptide libraries, on the detection of stress markers in saliva of pigs was evaluated in this study. For this purpose, saliva samples collected before and after the application of an acute stress model consisting of nasal restraining in pigs were processed without any treatment and with the two different treatments mentioned above. Protein separation by two-dimensional gel electrophoresis (2-DE) followed by identification of proteins using MALDI-TOF/TOF mass spectrometry (MS) was used as proteomic technique.ResultsThe application of each of the two different sample treatment protocols allowed the identification of unique proteins that could be potential salivary acute stress markers in pigs: lipocalin 1, protein S100-A8 and immunoglobulin M by enrichment of glycoproteins; protein S100-A9, double headed protease inhibitor submandibular gland, and haemoglobin by dynamic range compression; and protein S100-A12 by both protocols. Salivary lipocalin, prolactin inducible protein, light chain of immunoglobulins, adenosine deaminase and carbonic anhydrase VI were identified as potential markers in untreated saliva as well as one of the other treatments.ConclusionThe use of different procedures allowed the detection of different potential stress markers. Although from a practical point of view, the use of saliva without further treatment as well as the enrichment of glycoproteins are less expensive and easy to do procedures.

Proteomic Changes Between Populus Allotriploids and Diploids Revealed Using an iTRAQ-based Quantitative Approach.

Background: Polyploid breeding is a powerful approach for Populus genetic improve-ment because polyploid trees have valuable characteristics, including better timber quality and a higher degree of stress resistance compared with their full-sib diploids. However, the genetic mech-anism underlying this phenomenon remains unknown.Objective: To better understand the proteomic changes between Populus allotriploids and diploids, we examined the proteomic profiles of allotriploid and diploid Populus by iTRAQ labeling coupled with two-dimensional liquid chromatography and MALDI-TOF/TOF mass spectrometry (MS).Method: iTRAQ labeling coupled with two-dimensional liquid chromatography and MALDI-TOF/TOF mass spectrometry (MS).Results: Between the Populus allotriploid and the full-sib diploid, 932 differentially expressed proteins (DEPs) were identified. These DEPs were primarily involved in stress, defense, transportation, transcriptional and/or translational modification, and energy production. The pathway analysis indi-cated that most of the DEPs were implicated in carbohydrate transport and metabolism, nitrogen me-tabolism and glycolysis, and the ribosome assembly pathway. These data suggest high protein di-vergence between Populus allotriploids and diploids, and rapid changes during hybridization.Conclusion: The results provide new data for further understanding of the mechanisms of polyploid trees that generally display increased height growth compared with their full-sib diploids.

Identification of calmodulin binding proteins in the entomopathogenic fungus Beauveria bassiana.

Calmodulin (CaM) is a primary Ca2+ receptor and plays a pivotal role in a variety of cellular responses in eukaryotes. Even though a large number of CaM-binding proteins are well known in yeast, plants, and animals, little is known regarding CaM-targeted proteins in filamentous fungi. To identify CaM-binding proteins in filamentous fungi, we used a proteomics method coupled with co-immunoprecipitation (CoIP) and MALDI-TOF/TOF mass spectrometry (MS) in Beauveria bassiana. Through this method, we identified ten CaM-binding proteins in B. bassiana. One of the CaM-targeted proteins was the heat shock protein 70 (BbHSP70) in B. bassiana. Our biochemical study showed that ATP inhibits the molecular interaction between BbHSP70 and CaM, suggesting a regulatory mechanism between CaM and ATP for regulating BbHSP70.

Differential protein expression during sperm maturation and capacitation in an hermaphroditic bivalve,Pecten maximus(Linnaeus, 1758)

In order to investigate the mechanisms of final maturation and capacitation of spermatozoa in Pecten maximus, we used a 2D proteomic approach coupled with MALDI-TOF/TOF mass spectrometry (MS) and bioinformatics search against the Pecten database, to set up a reference map of the proteome of spawned spermatozoa, and identified 133 proteins on the basis of the EST database. These proteins are mainly involved in energy production, ion and electron transport (44%), cell movement (22%) and developmental processes (10%). Comparison between proteomes of spermatozoa collected before and after transit through the genital ducts of P. maximus led to the identification of differentially expressed proteins. Most of them are associated with energy metabolism (aconitate hydratase, malate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase), indicating important modifications of energy production during transit in gonoducts, potentially linked with acquisition of sperm motility. Three proteins involved in cell movement (Tektin-2, tubulin and microtubule-associated protein RP/EB family member 3) were down-regulated in spermatozoa stripped from the gonad. 40S ribosomal protein SA, involved in maturation of 40S ribosomal subunits, was also found to be down-regulated in spermatozoa obtained by induced spawning, suggesting reduction of the efficiency of RNA translation, a characteristic of late spermatozoon differentiation. These results confirm that maturation processes of P. maximus spermatozoa during transit through the gonoduct involve RNA translation, energy metabolism and structural proteins implicated in cell movement. Spermatozoa maturation processes clearly differ between P. maximus and gonochoric or alternately hermaphroditic bivalves, potentially in relation to reproductive strategies: the final maturation of the spermatozoon along the genital tract probably contributes to reduction of autofertilization in this simultaneously hermaphroditic species.

Identification and characterization of an 18.4kDa antimicrobial truncation from shrimp Litopenaeus vannamei hemocyanin upon Vibrio parahaemolyticus infection.

Hemocyanin (HMC) is a multifunctional protein which plays many essential roles in invertebrate organism. Recently more and more immune-related functions have been discovered on this protein. Here the shrimp was infected with Vibrio parahaemolyticus and the shrimp sera were analyzed by two-dimensional gel electrophoresis. Totally 15 spots were identified as significantly up-regulated spots and further analyzed by MALDI-TOF/TOF mass spectrometry (MS). Four of them were identified as HMC derived truncations (HMCS1, HMCS3, HMCS4 and HMCS5). The HMCS4 primary sequence was further determined via Edman N terminal sequencing, MALDI-TOF MS and amino acid sequence alignment. The result indicated that the HMCS4 was a 165aa fragment from shrimp HMC small subunit C-terminal. The HMCS4 immunological activities were further analyzed by agglutination experiment and antibacterial assay invitro. The results showed that the recombinant HMCS4 (rHMCS4) had strong agglutination and antibacterial activities against pathogenic bacteria at the optimum bacteriostasis concentration. In addition, the HMCS4 immunological activities were explored via mortality assay invivo. The shrimp was challenged with V. parahaemolyticus and rHMCS4 V. parahaemolyticus mixture separately. The shrimp mortality rate was significantly decreased at 96h post-infection with rHMCS4 injection. Our data showed that shrimp HMC truncation generation upon infection was an effective immune response against invaded pathogens. Moreover, these findings may have some potential applications in shrimp industry.

Proteomic analysis in the induction of nodular cluster cultures in the bromeliad Vriesea reitzii Leme and Costa

Nodular cluster cultures (NCs) are globular organogenic clumps with a high regenerative potential applied to the large-scale micropropagation of bromeliads. In the present work, we identified differentially expressed proteins involved in the induction of NCs from seeds and leaf explants of the Brazilian native bromeliad Vriesea reitzii. Those explants were inoculated into Murashige and Skoog (MS) liquid medium free of plant growth regulators (PGR). To promote the induction of NCs, the seeds were grown in MS medium supplemented with 4 μM α-naphthaleneacetic acid (NAA), and the leaf segments in MS medium supplemented with 4 μM NAA and 2 μM 6-benzylaminopurine (BAP). After 21 days in culture, samples of each type of explant were collected for histological analysis and protein extraction. Proteomic analysis was performed by two-dimensional (2D) electrophoresis and protein identification by MALDI-TOF–TOF mass spectrometry. Enhanced protein content and number of detected spots on cultures supplemented with PGR were observed as compared to the cultures maintained in PGR-free MS culture medium. Five differentially expressed proteins were identified during the induction of NCs: heat shock 22 kDa, chaperone protein dnaJ 50, S-adenosylmethionine synthase 3, UDP-arabinopyranose mutase 1, and 14-3-3-like protein E. Such proteins are involved in stress response, cell metabolism, and cell division. The ability to regulate the effects of stress conditions in which the explants were subjected shows the presence of competent tissues for the acquisition of the morphogenic route associated to the induction of NCs.

Secretome analysis of rice suspension-cultured cells infected by Xanthomonas oryzae pv.oryza (Xoo).

BackgroundRice bacterial blight (BB) caused by Xanthomonas oryzae pv.oryzae (Xoo) is one of the most devastating bacterial diseases in rice-growing regions worldwide. The rice-Xoo interaction is a classical model for studying the interaction between plants and pathogens. Secreted proteins play important roles in plant-bacterial interactions, but are poorly studied in the rice-Xoo system. Rice cv. Nipponbare is highly susceptible to Xoo. Here, we used two-dimensional difference gel electrophoresis (2D-DIGE) coupled with MALDI-TOF/TOF mass spectrometry (MS), to investigate secreted proteins in Nipponbare embryo cell suspension culture infected by Xoo.ResultsA total of 32 protein spots changed significantly (p < 0.05) by more than 1.5 fold in gel intensity after Xoo inoculation, and were identified by MS. They represent protein products of 11 unique genes, seven from rice and four from Xoo. Of the rice proteins, six up-regulated proteins are involved in cell wall modification, the TCA cycle, glycolysis and redox, while a down-regulated protein, CHIT16, is involved in plant defense. Quantitative Real-Time PCR showed that transcript levels were not correlated with secreted protein levels. Of the Xoo proteins, three of them were possibly located in the extracellular space as shown by transient expression assays in rice protoplasts. Two of the Xoo proteins were previously reported to be likely involved in pathogenicity, and the third gene, Xoo3654, is likely a negative regulator of Xoo virulence as its overexpression reduced Xoo pathogenicity in our study.ConclusionAmong the secreted proteins that responded to Xoo inoculation, we identified rice proteins involved in cell defense and Xoo proteins involved in pathogenicity. Our study also showed that Xoo3654 (X2) protein is likely a novel negative regulator of Xoo virulence. These results not only help us better understand the interaction between susceptible rice and Xoo, but also serve as a reference for studying the interaction between other plants and their pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-016-0091-z) contains supplementary material, which is available to authorized users.

Proteins involved in responses to biofilm and planktonic modes in Cronobacter sakazakii

Biofilms on surfaces of equipment and environments may be the major source of foodborne pathogens contamination in food. In this study, thirty-one Cronobacter sakazakii were identified by a combination of chromogenic agar, API-20e, and PCR targeting gluA and ropB genes. Furthermore, the highest amount and mature biofilm was formed by one typical isolate (Csa26) after 72 h using crystal violet staining (CVS) and scanning electron microscope (SEM) respectively. Thereafter, the two-dimensional gel electrophoresis (2-DE) technology was firstly applied to study the differential expression proteins between biofilm and planktonic cells. Using the MALDI/TOF/TOF mass spectrometry (MS) analysis combined with MASCOT research results, twenty-eight differential expression proteins were successfully identified at the threshold of 2.0 fold changes. In addition, the expression abundance of 13 proteins was expressed only or increased in biofilm cells. The findings presented here firstly reveal potential factors involved in biofilm formation at the proteomics level and provide new knowledge for revealing the biofilm-forming mechanism of C. sakazakii.

Development of a protein marker panel for characterization of human induced pluripotent stem cells (hiPSCs) using global quantitative proteome analysis

The emergence of new methods for reprogramming of adult somatic cells into induced pluripotent stem cells (iPSC) led to the development of new approaches in drug discovery and regenerative medicine. Investigation of the molecular mechanisms underlying the self-renewal, expansion and differentiation of human iPSC (hiPSC) should lead to improvements in the manufacture of safe and reliable cell therapy products. The goal of our study was qualitative and quantitative proteomic characterizations of hiPSC by means of electrospray ionization (ESI)-MSe and MALDI-TOF/TOF mass spectrometry (MS). Proteomes of hiPSCs of different somatic origins: fibroblasts and peripheral blood CD34+ cells, reprogrammed by the same technique, were compared with the original somatic cells and hESC. Quantitative proteomic comparison revealed approximately 220 proteins commonly up-regulated in all three pluripotent stem cell lines compared to the primary cells. Expression of 21 proteins previously reported as pluripotency markers was up-regulated in both hiPSCs (8 were confirmed by Western blot). A number of novel candidate marker proteins with the highest fold-change difference between hiPSCs/hESC and somatic cells discovered by MS were confirmed by Western blot. A panel of 22 candidate marker proteins of hiPSC was developed and expression of these proteins was confirmed in 8 additional hiPSC lines.

Characterization of potentially probiotic lactic acid bacteria isolated from olives: Evaluation of short chain fatty acids production and analysis of the extracellular proteome

Probiotic strains can exert positive effects on human health by various mechanisms, among which the production of short chain fatty acids (SCFA). All the SCFA, mainly acetic, propionic and butyric acid, display beneficial effects on human health; butyric acid is the most interesting for its role in the prevention and treatment of colonic diseases.In this study the ability of 17 potentially probiotic food-isolated lactic acid bacteria to produce SCFA, directly or indirectly through the production of lactic acid, was investigated. Propionic and butyric acids were quantified by gas chromatography; acetic and lactic acids were quantified by specific enzymatic kits. All the tested strains displayed the ability to produce significant amounts of acetic and lactic acids (in the range of g/L) and just small amounts of propionic and butyric acids (in the range of mg/L).The extracellular proteomes of two of these strains, Lactobacillus plantarum S11T3E and Lactobacillus pentosus S3T60C, were evaluated by coupling 2-DE and MALDI TOF-TOF mass spectrometry. This is an interesting approach to investigate a probiotic strain, since secreted proteins represent the first contact point between bacteria and the host after ingestion. Six and seven proteins, in different isoforms, were identified from L. pentosus S3T60C and L. plantarum S11T3E, respectively. All of them have a predicted extracellular location, indicating the effectiveness of the used protocol. L. plantarum S11T3E secretes several proteins with adhesive function, suggesting that in this strain the ability to adhere to gut mucosa depends on this kind of molecules. In L. pentosus S3T60C just one adhesive protein is secreted suggesting that other families of molecules play a role in its adhesive ability.

Proteomic-based comparison between populations of the Great Scallop, Pecten maximus

Comparing populations residing in contrasting environments is an efficient way to decipher how organisms modulate their physiology. Here we present the proteomic signatures of two populations in a non-model marine species, the great scallop Pecten maximus, living in the northern (Hordaland, Norway) and in the center (Brest, France) of this species' latitudinal distribution range. The results showed 38 protein spots significantly differentially accumulated in mantle tissues between the two populations. We could unambiguously identify 11 of the protein spots by Maldi TOF-TOF mass spectrometry. Eight proteins corresponded to different isoforms of actin, two were identified as filamin, another protein related to the cytoskeleton structure, and one was the protease elastase. Our results suggest that scallops from the two populations assayed may modulate their cytoskeleton structures through regulation of intracellular pools of actin and filamin isoforms to better adapt to their environment. Marine mollusks are non-model organisms that have been poorly studied at the proteomic level, and this article is the first studying the great scallop (P. maximus) at this level. Furthermore, it addresses population proteomics, a new promising field, especially in environmental sciences. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Comparison of Protein Changes in the Leaves of Two Bread Wheat Cultivars with Different Sensitivity under Salt Stress

Aims: Wheat performance under both Irrigated and dry land areas is affected by salinity and decreased. Like other plants, wheat cultivars try to indicate salt resistance through biochemical, physiological and morphological changes. In this study, responsive proteins in wheat leaves under salt stress were investigated by proteomics technique. Study Design: Factorial experiment based on a randomized complete block design Methodology: In this research, tolerant (Roshan) and sensitive (Ghods) cultivars were treated by 200mM salinity during 17 days. A proteomic technique was used to study proteins of leaf. Proteins of the leaves were extracted by TCA–acetone, and separated by two-dimensional gel electrophoresis at pH 4–7. Results: 200 repeatable spots were identified by using Melanie software and analyzed statically. 24 and 12 responsive spots were identified by using MALDI TOF-TOF mass spectrometry in tolerant and sensitive cultivars respectively. 5 responsive proteins were overlap between both cultivars. In tolerant cultivar, Expression of 19 spots was increased and expression of 5 spots was decreased. All the identified proteins are involved in the regulation of carbohydrate, energy and nitrogen metabolism, photosynthesis related Original Research Article Annual Research & Review in Biology, 4(11): 1784-1797, 2014 1785 proteins, ROS scavenging proteins, signaling related protein, transporters and chaperon. But, all identified proteins except one of them in sensitive cultivar were down regulated under salt stress. Conclusion: Based on this study, salt tolerant cultivar could alleviate salt stress. But salt sensitive cultivar couldn’t tolerate salinity and it seems that salt stress have destructive effects on its proteins. In other words, protein compositions of salt sensitive cultivar are more deeply damaged by salt stress than salt tolerant cultivar and protein decay could be a marker of the sensitivity of the concerned cultivar.

Study of Isobaric Interference in Quantification of Citrulline by Parallel Fragmentation Monitoring.

Parallel Fragmentation Monitoring (PFM), which is an analogue of selected reaction monitoring (SRM), is a recently developed method for quantification of small molecules by MALDI-TOF/TOF mass spectrometry (MS). It is well known that isobaric interference substances can be occasionally present in complex biological samples, and affect the accuracy of measurement by SRM. Unfortunately, by design it is not possible to assess whether isobaric interference happens in a SRM analysis. In contrast, the unique design of PFM should allow quick inspection for isobaric interference and subsequent correction. In this study, using arginine as an example, interference effect of isobaric structural analogs on the quantification of citrulline by PFM was evaluated. Our results showed that the presence of arginine affected the measured concentrations of citrulline standard solutions in a concentration dependent manner. Such interference could be observed readily in the MS/MS spectra, and contributed by [arginine+H-NH3](+) fragment ion. Because of having highly similar mass, (13)C-isotope of [arginine+H-NH3](+) fragment ion overlapped with monoisotope of [citrulline+H-NH3](+) fragment ion, which was used as the report ion for quantification. However, such interference could be mathematically eliminated or minimized through estimation of the signal intensity of the (13)C-isotopic peak of [arginine+H-NH3](+) from the intensity of the corresponding monoisotopic peak according to isotope distribution of elements. Furthermore, the presence of interfering fragment ions could be avoided by optimizing MALDI ionization condition. In conclusion, isobaric interference can happen in PFM, but can be easily identified in the mass spectra and eliminated (minimized) with simple methods.