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Thioredoxins are ubiquous proteins with 2 cysteines at the active site. The isoform 1 of Thioredoxin from Saccharomyces cerevisae (Trx1) has six sulphur aminoacids, two cysteines and four methionines. In this work we performed the replacement of cysteines by selenocysteines by growth of a transformed celular expression vector E. coli BL21-DE3 in selenocysteine containing culture medium. The Maldi-TOF spectra of Seleno/Sulphur substituted Trx1 revealed six component peaks with 46-48 Da range between them, that is the isotopic Seleno-Sulfur difference, showing the replacement of the Cysteines and Methionines to Selenocysteines and Selenomethionines. The Maldi-TOF spectra of the peptides derived from Trypsin digestion of the purified Thioredoxin (peptide mass fingerprint) show Selenocysteine and Selenomethionine containing peptides. Therefore we are demonstrating that cystein can be replaced by selenocystein and be metabolically converted to selenomethionine during Trx1 heterologous translation. Furthermore, the Maldi-TOF spectra are showing the presence of the most abundant isotopes of selenium inserted in the peptides containing cysteine and methionine, derived from the Trx1 digestion. The one dimensional 77Se–1H heteronuclear multiple quantum coherence NMR spectroscopy (1D-HMQC) for reduced Seleno substituted Trx1 (Se-Trx1), revealed three ressonance lines for 1Hβ1 from Selenocysteines 30 and 33, between 1.6 and 2,0 ppm. The bidimensional HMQC spectra (2D-HMQC) of the reduced Se-Trx1 show the 77 Se ressonance signal in 178 ppm, coupled with 1Hβ1 and 1Hβ2 lines between 2.1 and 1.8 ppm. The 1D-HMQC for oxidized Trx1 revealed the only one broad resonance in 2.6 ppm probably relative to the 1Hβ1 prótons. The 2D-HMQC spectrum of oxidized protein shows a higher chemical shift of selenocysteine 77Se (832 ppm) if compared to reduced state (178 ppm). Together these data are showing that the protocol of Se – S substitution developed here is a efficient method to label the active site of Thioredoxin 1 with a broad band chemical shift atom 77Se. Furthermore the large spectral window of the 77Se NMR detected between reduced and oxidized states of the Thioredoxin 1 shows that this atom is an excellent probe for accessing oxidative states and probably the conformational dynamics of the active site of the Se-Trx1.

An upper-division undergraduate- or graduate-level laboratory experiment, challenging students to identify bovine serum albumin, equine myoglobin, bovine calmodulin, and bovine cytochrome c, uses four methods of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): linear positive, in-source decay, peptide mass fingerprint, and postsource decay. Individual hands-on experience with each method is provided in a three week experiment. The importance of sequencing proteins/peptides when identifying proteomic targets using MALDI-TOF MS is emphasized.

We describe a simplified approach for the purification and characterization of urinary albumin, a key biomarker currently used for understanding the onset and prognosis of microalbuminuria. Urinary albumin was purified from human urine collected from diabetic kidney disease patients by using 2-stage tangential flow filtration process and set of column chromatography steps. The relative molecular mass of urinary albumin is 66,871 Da (SYNAPT G2 High Definition Mass Spectrometry System). Isolated urinary albumin was analyzed by SDS-PAGE, Western blotting, immunoelectrophoresis, Ouchterlony double-immunodiffusion, single radial immunodiffusion, size-exclusion HPLC and peptide mass fingerprint analysis. The size-exclusion HPLC elution profile of the purified urinary albumin was similar to that of a reference form of native albumin. Peptide mass fingerprint analysis of the purified urinary albumin yielded peptides that partially matched with known sequence of ALBU_HUMAN (P02768). This is the first report of purification and validation of immunochemically reactive form of urinary albumin from a large volume of urine of diabetic kidney disease patients. In this purification approach, the cost of the purified albumin is significantly lower.

In this study, GST Pull-down and mass spectrometry was applied to the precipitation and identification of the small GTP-binding protein (Rab7) interacting protein in hemocyte of Litopenaeus vannamei. According to the search in GenBank with the peptide mass fingerprint, the 45 kDa protein which was pulled down with the GST-tagged Rab7 (GST-Rab7, GTP bound form) was identified to be β-actin with 28% coverage of amino acid sequences. The interaction of Rab7 with β-actin was verified by both GST Pull-down and ELISA in vitro. Meanwhile, confocal microscopic observation showed that Rab7 could be co-localized with β-actin in hemocytes at 12 h post white spot syndrome virus (WSSV) infection (hpi). GST Pull-down and western blotting were used to analyze the cross-interaction between WSSV VP28, Rab7 and β-actin. The results showed that the GST-VP28, His-tagged Rab7 (His-Rab7) and His-β-actin formed a tripartite complex. At 12 hpi, confocal microscopic observation showed that WSSV could be co-localized with Rab7 and β-actin in hemocytes respectively. Furthermore, based on the in vivo neutralization assay, recombinant His-β-actin accelerated the infection of WSSV, conversely, recombinant His-Rab7 delayed WSSV infection in shrimp. These results suggested the interaction of Rab7 with β-actin and this interaction was involved in WSSV infection.

Spatial proteomics has the potential to significantly advance our understanding of biology, physiology and medicine. Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a powerful tool in the spatial proteomics field, enabling direct detection and registration of protein abundance and distribution across tissues. MALDI-MSI preserves spatial distribution and histology allowing unbiased analysis of complex, heterogeneous tissues. However, MALDI-MSI faces the challenge of simultaneous peptide quantification and identification. To overcome this, we develop and validate HIT-MAP (High-resolution Informatics Toolbox in MALDI-MSI Proteomics), an open-source bioinformatics workflow using peptide mass fingerprint analysis and a dual scoring system to computationally assign peptide and protein annotations to high mass resolution MSI datasets and generate customisable spatial distribution maps. HIT-MAP will be a valuable resource for the spatial proteomics community for analysing newly generated and retrospective datasets, enabling robust peptide and protein annotation and visualisation in a wide array of normal and disease contexts., MALDI-mass spectrometry imaging (MSI) can reveal the distribution of proteins in tissues but tools for protein identification and annotation are sparse. Here, the authors develop an open-source bioinformatic workflow for false discovery rate-controlled protein annotation and spatial mapping from MALDI-MSI data.

An untargeted shot-gun approach is described for the ultra-high-resolution analysis of fennel proteins by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) combined with a home-made Matlab search algorithm. The first step of the proposed bioinformatic strategy was the development of a custom-made fennel protein database, starting from the well-known, on-line available, protein NCBI database, under Foeniculum Vulgare organism, consisting of 231 total proteins. Partial and redundant forms of proteins, repeatedly included in the official NCBI database under different codes, were removed. In the final custom-made database, in addition to the 92 fennel specific non-redundant proteins, 10 proteins belonging to recognized allergenic sources associated with spice-mugwort-allergy syndrome (celery, carrot, parsley, birch, and mugwort) were also included. The second step was the in-silico enzymatic digestion, performed on all the 102 proteins, to obtain a theoretical list of m/z dataset of tryptic peptides. The Matlab processing data was the third and crucial step, necessary to search for in-silico mass calculated peptide sequences in the high resolution ICR mass spectra of the digested fennel extract. The final step was based on database searching in Peptide Mass Fingerprint (PMF) mode by using the matched m/z values as input data. The PMF search results confirmed the presence of 70 proteins (61 fennel specific and 9 allergenic proteins) inside the fennel extract.

Background: Streptococcus mutans (S. mutans) bacteria mainly cause dental caries in children. These bacteria are not considered oral indigenous bacteria since they are transmitted from people around children during their deciduous teeth eruption. The detection of these bacteria can be used for dental caries prevention in children. Purpose: To determine the strain and serotype of S. mutans by using matrix assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) and polymerase chain reaction (PCR) on dental plaque samples taken from mother-child pairs. Methods: Sixteen dental plaque samples of mother-child pairs were cultured on brain heart infusion broth (BHIB) and mitis salivarius bacitracin (MSB) media until S. mutans colony isolates were obtained. Next, the isolates of S. mutans colony were introduced into the target plates of MALDI-TOF MS, and then ionized to become peptide mass fingerprint (PMF). Afterwards, the colony isolates were detected by database software. The detected S. mutans DNA then was extracted by using conventional 727 bp PCR (serotype C). Results: Six strains of S. mutans were detected by MALDI-TOF MS method. Five samples were classified into UA159, two samples were 3SN1, two samples were NFSM1, two samples were 11A1, two samples were U138, two samples were 4SM1, and one sample was classified into another bacterium. Five out of 16 samples were detected by PCR as serotype C (UA159). Conclusion: Six strains of S. mutans were detected, namely UA159, 3SN1, NFSM1, 11A1, U138, and 4SM1, one of them (UA159) was detected as serotype C.

Isotope analyses are some of the most common analytical methods applied to ancient bone, aiding the interpretation of past diets and chronology. For this, the evaluation of “collagen yield” (as defined in radiocarbon dating and stable isotope research) is a routine step that allows for the selection of specimens that are deemed adequate for subsequent analyses, with samples containing less than ∼1% “collagen yield” normally being used for isotopic analysis but discounted for radiocarbon dating. The aims of this study were to use proteomic methods of MALDI–TOF (matrix assisted laser desorption ionization time-of-fligh mass spectrometry) and LC−ESI−MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) to investigate the endogeneity of the dominant proteinaceous biomolecules within samples that are typically considered to contain poorly preserved protein. Taking 29 archaeological samples, we evaluated the proteome variability between different acid-soluble fractions removed prior to protein gelatinization and considered waste as part of the radiocarbon dating process. We then correlated these proteomes against the commonly used “collagen yield” proxy for preservation. We found that these waste fractions contained a significant amount of both collagenous and noncollagenous proteins (NCPs) but that the abundance of these was not correlated with the acquired “collagen yield”. Rather than a depleted protein load as would be expected from a low “collagen yield”, the variety of the extracted NCPs was comparable with that commonly obtained from ancient samples and included informative proteins useful for species identification, phylogenetic studies, and potentially even for isotopic analyses, given further method developments. Additionally, we did not observe any correlation between “collagen yield” and peptide mass fingerprint success or between the different fractions taken from the same sample but at different radiocarbon pretreatment stages. Overall, these findings highlight the value in retaining and analyzing sample fractions that are otherwise discarded as waste during the radiocarbon dating process but more importantly, that low “collagen yield” specimens that are often misinterpreted by archaeologists as being devoid of protein can still yield useful molecular sequence-based information.

A rapid shot-gun method by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is proposed for the characterization of fennel proteins. After enzymatic digestion with trypsin, few microliters of extract were analyzed by direct infusion in positive ion mode. A custom-made non-redundant fennel-specific proteome database was derived from the well-known NCBI database; additional proteins belonging to recognized allergenic sources (celery, carrot, parsley, birch, and mugwort) were also included in our database, since patients hypersensitive to these plants could also suffer from fennel allergy. The peptide sequence of each protein from that derived list was theoretically sequenced to produce calculated m/z lists of possible m/z ions after tryptic digestions. Then, by using a home-made Matlab algorithm, those lists were matched with the experimental FT-ICR mass spectrum of the fennel peptide mixture. Finally, Peptide Mass Fingerprint searches confirmed the presence of the matched proteins inside the fennel extract with a total of 70 proteins (61 fennel specific and 9 allergenic proteins).

We attempted to study the antibacterial activity of rhizospheric Bacillus spp., to curb the bacterial blight of anthurium caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad). Twenty-eight bacterial isolates from rhizospheric regions were identified as different Bacillus spp. and Ochrobactrum sp. using 16S rRNA gene sequencing. B. subtilis BIO3 effectively inhibited the growth of Xad up to 1450.7 mm2, and extracted volatile organic metabolites from the isolate BIO3 inhibited the growth of Xad up to 1024 mm2. Tritrophic interaction of anthurium leaves bacterized with B. subtilis BIO3 and challenged with Xad resulted in the expression of 12 unique proteins compared to untreated control. Mascot Peptide Mass Fingerprint-based identification indicated that one was glutathione peroxidase, involved in defence mechanism, other six proteins were identified as leghemoglobin II, CTP synthase-like, predicted protein (Physcomitrella patens), centromere-associated protein E, grain size protein, and five proteins were hypothetical proteins. Foliar application with 1% liquid formulations (108 CFU/ml) of B. subtilis BIO3 significantly suppressed the bacterial leaf blight of anthurium up to 78% over untreated control and also increased the stem length and flower yield.

10 pages; International audience; Molluscs were one of the most widely-used natural resources in the past, and their shells are abundant among archaeological findings. However, our knowledge of the variety of shells that were circulating in prehistoric times (and thus their socio-economic and cultural value) is scarce due to the difficulty of achieving taxonomic determination of fragmented and/or worked remains. This study aims to obtain molecular barcodes based on peptide mass fingerprints (PMFs) of intracrystalline proteins, in order to obtain shell identification. Palaeoproteomic applications on shells are challenging, due to low concentration of molluscan proteins and an incomplete understanding of their sequences. We explore different approaches for protein extraction from small-size samples (

Ovarian cancer (OC) is the most lethal among female reproductive system malignancies. Depending upon the stage at presentation, the five year survival ratio varies from ∼92 to ∼30%. The role of biomarkers in early cancer diagnosis, including OC, is well understood. In our previous study, through an initial screening, we have analyzed eleven proteins that exhibited differential expression in OC using two-dimensional gel electrophoresis (2D-GE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometric (MALDI-TOF MS) analysis. In continuation of our previous study, the present work describes analysis of twenty more proteins that showed aberrant expression in OC. Among these, six showed consistent significant deregulation in the OC false discovery rate [FDR ≤ 0.05]. Upon MS analysis, they were identified as vimentin, tubulin beta 2C chain, tubulin alpha 1C chain, actin cytoplasmic 2, apolipoprotein A-I, and collagen alpha 2(VI) chain [peptide mass fingerprint (PMF) score ≥ 79]. One of the differentially regulated proteins, tubulin beta 2C chain, was found to be significantly (fold change, 2.5) enhanced in OC. Verification by western blot and enzyme-linked immunosorbent assay (ELISA) demonstrated that the tubulin beta 2C chain may serve as a valuable marker for OC (ANOVA p < 0.0001). The assessment of the likely association of TBB2C with OC in a larger population will not only help in developing clinically useful biomarkers in the future but also improve our understanding of the progression of OC disease.

The ability of mass spectrometry for discrimination between protein and peptide masses which are unique to specific pathogens provides an accurate and fast method for the detection of different types of pathogens, especially viruses. Capsid proteins are specific to each virus and can be used as a biomarker for detection of this pathogen. On the other hand, single-chain variable fragment (scFv) antibodies have been recently used to enhance the accuracy of immunoassay techniques. So conjugation of mass spectrometry and scFv antibody provides a very accurate and fast method for the detection of viruses. In this report, for the first time, we have immobilized scFv antibody of fig mosaic virus (FMV) on the magnetic nanoparticles (MNPs) to extract the virus capsid protein from complex biological media and subsequently identified this protein through both its intact molecular mass and peptide mass fingerprint (PMF) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

We propose a new high-throughput ultrafast method for large-scale proteomics approaches by speeding up the classic filter aided sample preparation protocol, FASP, from overnight to 2.5 h. Thirty-six samples can be treated in 2.5 h, and the method is scalable to 96-well plate-based pipelines. After a modification of the FASP-tube, the steps of protein reduction, protein alkylation, and protein digestion of complex proteomes are done in just 5.25 min, each one under the effects of an ultrasonic field (7 cycles: 30 s on and 15 s off). The new method was compared to the standard overnight digestion FASP protocol, and no statistical differences were found for more than 92.4%, 92%, and 93.3% of the proteins identified by studying the proteome of E. coli, mouse brain, and mouse liver tissue samples, respectively. Furthermore, the successful relative label-free quantification of four spiked proteins in E. coli samples, BSA, β-lactoglobulin, α-casein, and α-lactalbumin, was achieved, using either the ultrasonic-based FASP protocol or the classic overnight one. The new US-FASP method matches the analytical minimalism rules as time, cost, sample requirement, reagent consumption, energy requirements, and production of waste products are reduced to a minimum while maintaining high sample throughput in a robust manner as all of the advantages of the filter aided sample preparation protocol are maintained.

We developed a method to identify proteins adsorbed on solid surfaces from a solution containing a complex mixture of proteins by using Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass (MALDI-ToF mass) spectroscopy. In the method, we performed all procedures of peptide mass fingerprint method including denaturation, reduction, alkylation, digestion, and spotting of matrix on substrates. The method enabled us to avoid artifacts of pipetting that could induce changes in the composition. We also developed an algorithm to identify the adsorbed proteins. In this work, we demonstrate the identification of proteins adsorbed on self-assembled monolayers (SAMs). Our results show that the composition of proteins on the SAMs critically depends on the terminal groups of the molecules constituting the SAMs, indicating that the competitive adsorption of protein molecules is largely affected by protein-surface interaction. The method introduced here can provide vital information to clarify the mechanism underlying the responses of cells and tissues to biomaterials.

Ants (Hymenoptera, Apocrita, Aculeata, Formicoidea) comprise a well-succeeded group of animals. Like bees and wasps, ants are mostly venomous, having a sting system to deliver a mixture of bioactive organic compounds and peptides. The predatory giant ant Dinoponera quadriceps belongs to the subfamily Ponerinae that includes one of the largest known ant species in the world. In the present study, low molecular weight compounds and peptides were identified by online peptide mass fingerprint. These include neuroactive biogenic amines (histamine, tyramine, and dopamine), monoamine alkaloid (phenethylamine), free amino acids (e.g. glutamic acid and proline), free thymidine, and cytosine. To the best of our knowledge, most of these components are described for the first time in an ant venom. Multifunctional dinoponeratoxin peptide variants (pilosulin- and ponericin-like peptides) were characterized that possess antimicrobial, hemolytic, and histamine-releasing properties. These venom components, particularly peptides, might synergistically contribute to the overall venom activity and toxicity, for immobilizing live prey, and for defending D. quadriceps against aggressors, predators, and potential microbial infection.

Pigment-binding proteins play important roles in crustacean shell colour change. In this study, a red colour-related pigment-binding protein, designated LvPBP75, was purified from the shell of Litopenaeus vannamei. HPLC and PAGE analysis showed that LvPBP75 was a homogeneous monomer with molecular mass of 75kDa. Peptide mass fingerprint analysis revealed that LvPBP75 belonged to hemocyanin, and the released pigment from heated LvPBP75 showed a λmax at 481nm in acetone. The significant red-colour change temperatures were detected at 30 and 80°C, respectively. Based on the determined amino acid fragments, a full-length cDNA of LvPBP75 was cloned and sequenced. The ORF encodes a protein of 662 amino acids having 80% identity with penaeidae hemocyanin. These results strongly suggest a novel function of hemocyanin, namely binding with pigment, and its involvement in L. vannamei shell colour change.

AimsDiagnostic ambiguities regarding the malignant potentiality of oral submucous fibrosis (OSF), an oral precancerous condition having dysplastic and non-dysplastic isoforms are the major failure for early intervention of oral squamous cell carcinoma (OSCC) patients. Our goal is to identify proteomic signatures from biopsies that can be used as precancer diagnostic marker for patient suffering from OSF.MethodsThe high throughput techniques adopting de novo peptide sequencing (1D SDS-PAGE coupled nanoLC MALDI tandem mass spectrometry (MS/MS)-based peptide mass fingerprint), immunohistochemistry (IHC), Western blot (WB) and real-time PCR (RT-PCR) analysis are considered for such biomarker identification and multilevel validations.ResultsAlpha-enolase is identified as an overexpressed protein in biopsies of oral submucous fibrosis with dysplasia (OSFWD) compared with oral submucous fibrosis without dysplasia (OSFWT) and normal oral mucosa (NOM). Total proteome analysis of an overexpressed protein band around 47 kDa of OSFWD identifies 334 peptides corresponding to 61 human proteins. Among them α-enolase is identified as a prime protein with highest number of peptides (44 out of 334 peptides) and sequence coverage (66.4%). Furthermore, RT-PCR, WB and IHC analysis also show mRNA and tissue level upregulation of α-enolase in OSFWD validating α-enolase as precancer marker.ConclusionsThis study for the first time identifies and validates α-enolase as a novel biomarker for early diagnosis of malignant potentiality of OSF. Hence, the identified protein marker, α-enolase can help in early therapeutic intervention of OSF patients leading to the reduction of patient’s pain, treatment cost and enhancement of patient’s quality of life.

RATIONALE Species identification of hair is routinely done by microscopic analysis. Following previous studies that used protein analysis to characterize species markers in hair and wool, the present work aims at covering a larger number of species and to ultimately offer a method for rapid hair identification in forensics and archaeology. METHODS Hair is mostly made of alpha-keratins; these proteins have only been sequenced in a handful of species and most animal families are under-represented. Using a methodology developed for the characterization of peptidic markers in tissues such as bone (peptide mass fingerprinting or PMF) and commonly applied on collagen, hair from common North American fur-bearing species was analyzed by MALDI-TOF-MS to obtain peptidic profiles. RESULTS Alpha-keratin peptides that are typically dominant on peptide mass profiles of hair were chosen as markers. Matching peaks were identified for each species tested and compared to known sequences from related organisms whenever possible. The markers were used to create a flowchart to narrow down identification to the family level. CONCLUSIONS The methodology was developed on a limited numbers of markers chosen for their variability and reliability on the peptide mass fingerprint. In the absence of genetic sequences, that strategy is a quick way to compare species from a common geographic origin. The work presented here was focused on North American species but could be applied to other animal families.

We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (λ-FLCs) and kappa free light chains (κ-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of λ-FLC and κ-FLC are 22,933.397 and 23,544.336Da respectively. Moreover, dimer forms of each FLC were also detected in mass spectrum which corresponds to 45,737.747 and 47,348.028Da respectively for λ-FLCs and κ-FLCs. Peptide mass fingerprint analysis of the purified λ-FLCs and κ-FLCs has yielded peptides that partially match with known light chain sequences viz., gi|218783338 and gi|48475432 respectively. The tryptic digestion profile of isolated FLCs infers the exclusive nature of them and they may be additive molecules in the dictionary of urinary proteins. This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing. This simple and cost-effective approach to purification of FLCs, together with the easy availability of urine samples make the large-scale production of FLCs possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

Saccharomonospora azurea SZMC 14600 is a member of the family Pseudonocardiaceae exclusively used for industrial scale production of primycin a large 36-membered non-polyene macrolide lactone antibiotic belonging to the polyketide class of natural products. Even though maximum antibiotic yield has been achieved by empirically optimized two-step fermentation process, little is known about the molecular components and mechanisms underlying the efficient antibiotic production. In order to identify differentially expressed proteins (DEPs) between the pre- and main-fermentation stages of primycin, comparative 2D-PAGE experiments were performed. In total, 98 DEP spots were reproducibly detected, out of which four spots were excised from gels, and identified through MALDI-TOF/TOF mass spectrometry. Peptide mass fingerprint analysis revealed peptide matches to HicB antitoxin for the HicAB toxin-antitoxin system (EHK86651), to a nucleoside diphosphate kinase regulator ((Ndk; EHK81899) and two other proteins with unknown function (EHK88946 and EHK86777).

Resistant (WL711+ Lr 57) and susceptible (WL711− Lr 57) near isogenic lines of wheat were used to unravel the molecular basis of seedling resistance conferred by Lr 57 gene during wheat- Puccinia triticina (race 77-5) interaction. Differentially expressed proteins during incompatible and compatible interactions were identified through 2D-PAGE and their peptide mass fingerprint was determined by MALDI TOF. In silico functional annotation revealed the presence of diverse categories of proteins associated with defense, photosynthesis, nucleotide repair, DNA replication, protein synthesis and transposable elements. For the first time, the differential expression of a nucleotide repair related protein, MMS19, during wheat- Puccinia interaction is being reported in this study. The proteomic analysis of 96 hpi WL711−Lr57 revealed the negative regulation of JA, SA and ET mediated pathways, thus accounting for its susceptibility. Transcriptomic validation was performed for a set of selected potential candidate proteins through qRT-PCR. The identified proteins possess the potential to be exploited in development of novel wheat varieties tolerant to leaf rust at seedling stage.

Protease mediated proteolysis has been widely implicated in virulence of necrotrophic fungal pathogens. This is counteracted in plants by evolving new and effective antimicrobial peptides (AMP) that constitute important components of innate immune system. Peptide extraction from rhizome of Zingiber zerumbet was optimized using ammonium sulphate (50–80% w/v) and acetone (60 and 100% v/v) with maximal protein recovery of 1.2 ± 0.4 mg/g obtained using 100% acetone. Evaluation of inhibitory potential of Z. zerumbet rhizome protein extract to prominent hydrolases of necrotrophic Pythium myriotylum revealed maximal inhibition of proteases (75.8%) compared to other hydrolytic enzymes. Protein was purified by Sephacryl S200HR resin resulting in twofold purification and protease inhibition of 84.4%. Non-reducing polyacrylamide gel electrophoresis (PAGE) of the fractions yielded two bands of 75 kDa and 25 kDa molecular size. Peptide mass fingerprint of the protein bands using matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectroscopy (MS) and subsequent MASCOT searches revealed peptide match to methylesterase from Arabidopsis thaliana (15%) and to hypothetical protein from Oryza sativa (98%) respectively. Further centrifugal filter purification using Amicon Ultra (10,000 MW cut-off) filter, yielded a prominent band of 25 kDa size. Concentration dependent inhibition of zoospore viability by Z. zerumbet AMP designated as ZzAMP was observed with maximal inhibition of 89.5% at 4 µg protein and an IC50 value of 0.59 µg. Studies are of particular relevance in the context of identifying the molecules involved in imparting below ground defense in Z. zerumbet as well in development of AMPs as potential candidate molecules for control of necrotrophic pathogens of agricultural relevance.

Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

Following the report of Mycobacterium tuberculosis proteins found in archaeological bone by Boros-Major et al. (2011), we attempted to identify M. tuberculosis proteins in mummified lung tissues from which ancient DNA success had already been reported. Using a filter-aided sample preparation protocol modified for ancient samples we applied shotgun proteomics to seven samples of mummified lung, chest and pleura tissues. However, we only identified four peptides with unique matches to the M. tuberculosis complex, none of which were unique to M. tuberculosis, although we did identify a range of human proteins and non-mycobacterial bacterial proteins. In light of these results, we question the validity of the peptide mass fingerprint (PMF) approach presented by Boros-Major et al. (2011), especially because the PMF spectrum presented in Boros-Major et al. (2011) has similarities to that of human collagen, the dominant protein in the tissue under investigation. We explore the challenges of using proteomic approaches to detect M. tuberculosis, and propose that, given the contentious outcomes that have plagued ancient protein research in the past, the susceptibility of ancient material to modern contamination, and the degradation inherent in archaeological samples, caution is needed in the acquisition, analysis and reporting of proteomic data from such material.

Wilms' tumors are one of the most common malignant, solid intra-abdominal tumors observed in children. Although potential tumor markers have been found, inflammatory cytokines interfere with the process of specific protein identification. The present study was undertaken to identify post-traumatic stress-related factors of Wilms' tumors and to verify the accuracy of early-stage tumor-specific serum protein markers. Serum samples were screened for differentially-expressed proteins using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). Potential markers were isolated and purified using solid-phase extraction (SPE) and SDS-PAGE. Following enzymatic digestion of the protein samples, the peptide fragments were detected with high performance liquid chromatography-mass spectrometry. The obtained peptide mass fingerprint was searched in the Swiss-Prot protein sequence database via the Mascot search engine. Differentially-expressed proteins were verified using western blot analysis. Differentially-expressed proteins with a mass/charge of 5,816 were screened out using SELDI-TOF-MS, and significant differences between the tumor and control groups, and the trauma and control groups were observed. Target proteins were isolated and purified using SPE and SDS-PAGE. Thioredoxin 1 (Trx1) was found to be differentially expressed. In the serum of children with Wilms' tumors, there was an increase in the level of the post-traumatic stress-related inflammatory factor, Trx1, as compared with the normal control group. Thus, the results of this study indicate that Trx1 presents a potential post-traumatic stress-related factor of Wilms' tumors.

Collagen XXVIII is the last discovered member of the collagen superfamily and thus has been only sparsely investigated. We studied collagen XXVIII in zebrafish to gain insight into its structure, evolution and expression. In contrast to human and mouse, the zebrafish genome contains four collagen XXVIII genes, col28a1a and - b , and col28a2a and - b . Genomic context and phylogenetic analysis revealed that the a2 branch was lost during evolution of mammals, whereas the duplication of the a1 and a2 branches results from the whole genome duplication in the teleost lineage. Sequence analysis revealed conservation of domain structure and the unique imperfections in the triple helical domain. Two major forms of collagen XXVIII were identified, Col28a1b in adult and Col28a2a in 3–5 dpf zebrafish. Composite agarose/polyacrylamide gel electrophoresis revealed that both these chains mainly form dimers of trimers, although Col28a1b appears to be more polydisperse. Homodimers are abundant, although it is possible that complexes consisting of Col28a2a and Col28a1a or -a2b occur. Peptide mass fingerprint analysis revealed that the C-terminal Kunitz domain is often proteolytically processed. In contrast to murine collagen XXVIII, the zebrafish orthologs are widely expressed and not only present in the nervous system. They are differentially expressed in the liver, thymus, muscle, intestine and skin. Altogether our results point to a unique nature of collagen XXVIII within the collagen family.

We describe a simplified approach for purification and characterization of human ‘IgG-Fc’ fragment used widely as immunochemical tool and for therapeutic purposes. The ‘Fc’ fragment was purified from human IgG in a 3-stage column chromatography. The purified ‘Fc’ fragment appeared as a dimer glycoprotein with an apparent molecular mass of 52,981 Dalton (Ultraflex MALDI TOF/TOF). The Size-exclusion HPLC profile of the purified ‘Fc’ fragment of human IgG matched that of a commercially procured reference ‘Fc’ fragment material. The purity of the ‘Fc’ fragments was >99% by SDS-PAGE and size-exclusion HPLC. The results of Western blotting, immunoelectrophoresis, and mass spectrometry analysis indicate a high purity of the ‘Fc’ fragment. Peptide mass fingerprint analysis of the purified ‘Fc’ protein yielded peptides that partially match the known database sequences of FCG3B_HUMAN (Uniprot ID: O75015). This method of purification of the ‘Fc’ fragment is suitable for achieving high purity level of ‘Fc’ fragment protein. With this purification approach, the cost of the purified ‘Fc’ fragment of human IgG is significantly reduced as compared with the current market price of IgG-Fc fragment protein in international market. The purified ‘IgG-Fc’ fragment protein was found to be negative for major viral markers.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC50 = 24 ng) and RsKI (IC50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.

The cystine-knot metallocarboxypeptidase inhibitors (MCPIs) are peptides that contribute to control proteolytic activity, involved in storage, growth and maintenance of plants. Lately studies reported several MCPIs with potential use in biomedical applications; as anti-cancer, anti-thrombotic, anti-malaric and anti-angiogenic agents. We report the isolation, purification, chemical stability and biochemical characterization of a novel carboxypeptidase A inhibitor (YBPCI) isolated from Capsicum annuum L. var. Yellow Bell Pepper, the first cystine-knot miniprotein (CKM) of the species. We demonstrate the stability of YBPCI (IC50 = 0.90 μg/ml) to high temperatures, high salt concentration and extreme pH values. MALDI-TOF/MS analysis detected a molecular weight of 4057 Da, and peptide mass fingerprint resulted in no matches with other protease inhibitors. In vitro gastrointestinal digestion subjecting YBPCI to pH 2 incubation and proteolytic attack resulted in complete inhibitory activity. To summarize, there are no reports to date of carboxypeptidase inhibitors in C. annuum species, giving our report much more relevance., Facultad de Ciencias Exactas, Centro de Investigación de Proteínas Vegetales

Open Access; International audience; We isolated from aerobic and anaerobic blood culture bottles from a febrile patient, a Helicobacter-like Gram negative, rod-shaped bacterium that MALDI-TOF MS failed to identify. Blood agar cultures incubated in a microaerobic atmosphere revealed a motile Gram negative rod, which was oxidase, catalase, nitrate reductase, esterase, and alkaline phosphatase positive. It grew at 42 • C with no detectable urease activity. Antimicrobial susceptibility testing showed that the organism was susceptible to beta-lactams, gentamicin, erythromycin, and tetracycline but resistant to ciprofloxacin. Electronic microscopy analysis revealed a 3 × 0.5 µm curved rod bacterium harboring two sheathed amphitrichous flagella. Whole genome sequencing revealed a genome 1,708,265 base-pairs long with a GC content of 37.80% and a total of 1,697 coding sequences. The genomic analyses using the nucleotide sequences of the 16S rRNA gene, hsp60 and gyrB genes, as well as the GyrA protein sequence, and the results of Average Nucleotide Identity and in silico DNA-DNA hybridization suggest evidence for a novel Helicobacter species close to Helicobacter equorum and belonging to the group of enterohepatic Helicobacter species. As soon as the particular peptide mass fingerprint of this pathogen is added to the spectral databases, MALDI-TOF MS technology will improve its identification from clinical specimens, especially in case of " sterile infection ". We propose to associate the present strain with the Latin name of the place of isolation; Caesarodunum (Tours, France) and suggest " Helicobacter caesarodunensis " for further description of this new bacterium.

A dimeric protease designated as EuP-82 was purified from Euphorbia cf. lactea latex. Since its proteolytic activity was inhibited by a serine protease specific inhibitor (PMSF), EuP-82 was classified as a serine protease. N-glycan deglycosylation tests revealed that EuP-82 was a glycosylated protein. MALDI-TOF MS showed that EuP-82 was a homodimer, which was its active form. The optimal conditions for fibrinogenolytic activity were at pH 11 and 35 °C. EuP-82 enzyme had broad range of pH stability from 4 to 12. Moreover, the enzyme was still active in the presence of reducing agent (β-mercaptoethanol). EuP-82 was a proline-rich enzyme (about 20.69 mol%). Increased proline production can be found in higher plants in response to both biotic and abiotic stresses, high proline in the molecule of EuP-82 might stabilize its activity, structure and folding. Based on the N-terminal amino acid sequences and peptide mass fingerprint (PMF) of EuP-82, the enzyme was identified as a new serine protease. The digested products from EuP-82 cleavage of human fibrinogen were analyzed by SDS-PAGE and PMF. The results confirmed that EuP-82 could digest all subunits of human fibrinogen. EuP-82 cleaved fibrinogen with a Michaelis constant (Km) of 3.30 ± 0.26 μM; a maximal velocity (Vmax) of 400.9 ± 0.85 ng min(-1); and a catalytic efficiency (Vmax/Km) of 121.5 ± 9.25 ng μM(-1) min(-1). EuP-82 has potential for use in medicinal treatment, for example thrombosis, since the enzyme had fibrinogenolytic activity and high stability.

BACKGROUND Pholiota nameko water-soluble protein (PNWSP), isolated from the dried fruiting bodies of Pholiota nameko, was purified by a successive chromatographic process using Q anion exchange column, SP cation exchange column and Superdex 200 gel filtration column. PNWSP was assessed for antioxidant activities in different assay systems, and the effect on cell proliferation of human breast cancer MCF7 cells was investigated. RESULTS Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analytical ultracentrifugation analysis revealed the homogeneity of PNWSP with apparent molecular weight of ∼43 kDa, and the identification was confirmed by peptide mass fingerprint analysis. It showed potential antioxidant activities in scavenging free radicals, reducing power and chelating effect on Fe2+, and had a protective effect against DNA amage. Moreover, it inhibited the proliferation of MCF7 cells by inducing apoptosis, in which the change in cell cycle distribution and the loss of mitochondrial transmembrane potential were observed by flow cytometry. ConcluSIONS The results showed that PNWSP could be a natural antioxidant and developed as a potential chemotherapeutic agent candidate against human breast cancer. © 2015 Society of Chemical Industry

ClbR is a Zn(II)2Cys6 transcriptional activator that controls the expression of cellulase-related genes in response to Avicel and cellobiose in Aspergillus aculeatus. A clbR-overexpressing strain (clbR-OE) that expresses the clbR gene at levels sevenfold higher than the control strain sustainably produced xylanolytic and cellulolytic activities during 10-day cultivation of A. aculeatus, enabling synchronization of xylanolytic and cellulolytic activities at a maximum level. However, clbR overexpression did not simultaneously increase levels of all xylanolytic and cellulolytic enzymes. Peptide mass fingerprint analysis revealed markedly increased production of FIa-xylanase in clbR-OE, whereas expression of FIII-avicelase and FII-carboxymethyl cellulase was unaffected and expression of hydrocellulase was lower in clbR-OE than in the control. Northern blot analysis confirmed that these effects of clbR overexpression on enzyme production were mediated at the transcriptional level. These data suggest that ClbR participates in diverse signaling pathways to control the expression of cellulosic biomass-degrading enzymes in A. aculeatus.

Graphical abstract Identified protein peaks (231, 232, 241, 251, 252, and 253) of cauliflower ‘H41’ plants by the ProteomeLab PF-2D Protein Fractionation System (Beckman Coulter) in comparing FH and NFC at 24 h., Highlights • Cauliflower cultivars H41 and H69 are tolerant to high temperature and flooding, respectively. • Physiological and proteomic responses of cauliflower genotypes to flood stress at 40 °C were determined. • Comparative proteomic analysis identified 85 protein peaks during different time periods in all genotypes. • Differentially regulated proteins predominantly functioned in photosynthesis and metabolism., High-temperature and waterlogging are major abiotic stresses that affect the yield and quality of cauliflower. Cauliflower cultivars ‘H41’ and ‘H69’ are tolerant to high temperature and flooding, respectively; however, ‘H71’ is sensitive to both stresses. The objectives of this study were to identify the proteins that were differentially regulated and the physiological changes that occurred during different time periods in ‘H41’, ‘H69’, and ‘H71’ when responding to treatments of flooding, 40 °C, and both stresses combined. Changes in the leaf proteome were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and identified by Mascot peptide mass fingerprint (PMF) and database searching. Stress treatments caused significant reductions in electrolyte leakage, chlorophyll fluorescence Fv/Fm, chlorophyll content, and water potential as stress times were prolonged. By the comparative proteomic analysis, 85 protein peaks that were differentially expressed in response to combination treatments at 0, 6, and 24 h, 69 (33 in ‘H41’, 29 in ‘H69’, and 9 in ‘H71’) were identified, of which were cultivar specific. Differentially regulated proteins predominantly functioned in photosynthesis and to a lesser extent in energy metabolism, cellular homeostasis, transcription and translation, signal transduction, and protein biosynthesis. This is the first report that utilizes proteomics to discover changes in the protein expression profile of cauliflower in response to heat and flooding.

To understand the effects of nanoparticles on microorganisms, we experimentally investigated the effects of CeO2 nanoparticles (CeNPs) on yeast (S. cerevisiae) focusing on microbial metabolites and intracellular proteins. The yeast were harvested from a yeast extract peptone dextrose medium containing 0, 10, 100, and 250 ppm of CeNPs and incubated for 120 h in 1 mM NaCl solution at three different pH values: 3, 5, and 7. The yeast released organic matter, P, K, and Mg into the NaCl solution at all pH values, even without CeNPs. Distinct differences were detected in the released organic species and intracellular proteins after exposure to CeNPs. High-performance liquid chromatography revealed that various organic species released from the yeast were expressed or suppressed after exposure to CeNPs. Although cytotoxicity was not caused by CeNPs, the results of the peptide mass fingerprint analysis of the intracellular protein revealed that Eno2p, a glycolysis enzyme, was expressed after exposure to CeNPs. These results suggest that nanoparticles have the potential to alter microbial metabolism, leading to changes in the compositions of the released substances in the surrounding environment.

To better understand the molecular pathogenesis of white spot syndrome virus (WSSV) inLitopenaeus vannamei(Boone, 1931), the protein expression profile in gills was characterized. Farmed shrimp WSSV positive were randomly sorted based on nested PCR. The proteomic analysis of gill proteins was performed using two-dimensional electrophoresis (2-DE), with isofocalisation on an immobilized linear gradient (pH 3-10), followed by separation based on molecular weight using 12.5% denaturating polyacrylamide gel electrophoresis (SDS-PAGE). The comparative analysis of the 2-DE profile between the two groups revealed eight differentially expressed spots in gills of naturally infected shrimp. The spots were successfully identified using MALDI-TOF mass spectrometry peptide mass fingerprint. The up-regulated proteins unique to infected shrimp were identified as peptidyl-prolyl isomerase, mortality factor 4-like protein 1, calreticulin, recombination activating protein, failed axon connection protein, 40S ribosomal S2 and N-deacetylase/N-sulfotransferase. The down-regulated protein unique to non-infected shrimp (control group) was identified as an inhibitor of apoptosis. The differentially expressed proteins are involved in several important cellular processes, such as host defence and protein metabolism. The present work contributes to a better understanding of the overall molecular responses elicited by WSSV infection inL. vannamei, as well as to point out potential molecular biomarkers to evaluate the susceptibility to the virus and the sanitary status in farmed shrimp.

Scope Hypertension is a risk factor for arteriosclerosis. In this study, we investigate the antihypertensive effect of protease-digested rice bran in a spontaneously hypertension rat (SHR) model. We also purify a novel antihypertensive peptide from the digest. Methods and results Thermolysin-digested rice bran (TRB) is administered to SHRs for 4 weeks, and systolic blood pressure (SBP) was measured weekly using the tail-cuff method. TRB shows an antihypertensive effect in a dose-dependent manner. TRB also reduces angiotensin I-converting enzyme (ACE) activity in lung tissue and serum troponin I levels. TRB is fractionated by HPLC and ACE-inhibitory activity in the HPLC fractions is measured. Peptides LRA and YY are identified from the two fractions with the strongest ACE-inhibitory activity. Amino acid sequence of these peptides are found in a vicilin-like seed storage protein, and identified in rice bran protein using the peptide mass fingerprint method. We confirm that LRA and YY are cleaved by thermolysin digestion of a model synthetic peptide. Orally administered LRA (0.25 mg kg-1 ) or YY (0.5 mg kg-1 ) lowers the SBP of SHRs at 4 h after administration. Conclusion We identify a novel, orally active antihypertensive peptide, LRA from the digest of rice bran protein.

Background Zinc transporter (tzn-1) of Neurospora crassa plays a crucial role in conidiation pathway, as its removal results in aconidiation which was reported in our earlier studies. Objectives The main objective of this study was to analyze the role of tzn-1 in conidiation process, by comparing knockout (KO) mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) with wild oak ridge (OR) 74 'A' strain by 'Proteo-genomic' approach. Methods To identify the commonly expressed protein spots in knockout (KO) mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) by comparing with wild oak ridge (OR) 74 'A' strain. Two sets (Δtzn-1 to wild and Δacon-3 to wild) were analyzed by combining 2- Dimensional gel electrophoresis (2DE) with Matrix Associated Laser Desortion/Ionization mass spectrometry -Peptide Mass Fingerprint (MALDI-PMF). Then, the peptide sequences which were obtained by MASCOT (database software) were identified by FGSC BLASTp search analysis. Finally, to evaluate the expression of the KO mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) in comparison to wild (OR) 74 'A' type was analyzed by Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) studies. Results 2DE and MALDI-PMF has shown the nine commonly overexpressed protein spots from the two sets (Δtzn-1 to wild and Δacon-3 to wild). Peptide sequences were obtained by MASCOT (database software) analysis and peptide sequences were identified by FGSC BLASTp search. Eight sequences have shown the similarities with the genes involved during the early stages of conidial and sexual development. Our qRT-PCR analysis has shown that tzn-1 gene was upregulated in contrast to acon-3 gene in absence of iron concentration and down regulated with increase in iron concentrations in wild samples. With increase in zinc supplements, the tzn-1 gene is normally regulated and shown contrasting feature in absence of zinc and acon-3 gene is normally regulated both in presence and absence of zinc. At regular time intervals, declined growth rate was observed after 18hours of induction. Conclusion Thus, we conclude that tzn-1 and acon-3 genes were actively participating in early stages of conidial process and metal ions play some crucial role in the development of the organism.

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from loach skin. The yields of ASC and PSC were 22.42% and 27.32%, respectively. Sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and peptide mass fingerprint analysis revealed that loach skin contained type I collagen. There were 212 imino acids/1000 residues in ASC and 193 imino acids/1000 residues in PSC. Fourier transform infrared spectrometry analysis, UV measurements and circular dichroism confirmed that loach skin collagen had a triple helical structure. The denaturation temperatures of ASC and PSC were 36.03°C and 33.61°C, respectively. Zeta potential analysis revealed that net zero charge values of ASC and PSC were 6.42 and 6.51, respectively. Therefore, loach skin collagen may be an alternative to terrestrial mammalian collagen and may enhance the added value of this fish species.

The identification of carbohydrate-protein interactions is central to our understanding of the roles of cell-surface carbohydrates (the glycocalyx), fundamental for cell-recognition events. Therefore, there is a need for fast high-throughput biochemical tools to capture the complexity of these biological interactions. Here, we describe a rapid method for qualitative label-free detection of carbohydrate-protein interactions on arrays of simple synthetic glycans, more complex natural glycosaminoglycans (GAG), and lectins/carbohydrate binding proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The platform can unequivocally identify proteins that are captured from either purified or complex sample mixtures, including biofluids. Identification of proteins bound to the functionalized array is achieved by analyzing either the intact protein mass or, after on-chip proteolytic digestion, the peptide mass fingerprint and/or tandem mass spectrometry of selected peptides, which can yield highly diagnostic sequence information. The platform described here should be a valuable addition to the limited analytical toolbox that is currently available for glycomics.

In Matrix Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI), identification of observed proteins remains a challenge primarily due to the drop in sensitivity of time-of-flight (TOF) mass spectrometers beyond the mass range of 25-35 kDa (Francese et al. High Throughput Screen 12: 156-174, 2009) and inadequate mass resolving power at those molecular weights. To counteract this, a bottom-up proteomic approach is often employed. Proteolysis digests proteins into smaller peptide fragments, typically between 500 and 3000 Da which are easier to detect with a higher mass accuracy. Here, we describe the addition of a MS-compatible detergent to the endopeptidase trypsin, to enhance in situ proteolysis efficiency and peptide intensity.

The aim of this study was to elucidate the changes of Proteomics in MG-63 cells after induced by calcitonin gene-related peptide (CGRP). The subcultured MG63 cells were randomly divided into CGRP group and the control group. Two-dimensional electrophoresis (2-DE), coomassie brilliant blue gel staining and mass spectrometry were used to analyze the changes of protein extracted from the two groups. Six protein spots with significant differences were selected to take in-gel digestion, peptide mass fingerprint analysis and IPI database search. There were more than 967±17 protein spots separated by Two-dimensional gel electrophoresis and the matching rate was (85.1±1.4%). Compared with the control group, six protein spots were significantly different in CGRP-induced group and expression of the 6 differently proteins was downregulated. Mass spectrometry analysis identified 5 proteins, including ribosome binding protein p180 which is related to new synthetic protein's translocation and calcium-binding protein (HRNR Hornerin) that can affect intracellular calcium concentration to regulate cell activity. These changes of proteome, including several groups of proteins that influence calcium ion concentration and new protein translocation, suggested that CGRP may regulate the cells by the second messengers and cytokines. This study contributes to a better understanding of the molecular mechanisms in CGRP-induced MG-63 cells.

The research reported in this paper was conducted to analyze erythrocyte membrane associated proteins (ERMBPs) of some of chronic myeloid leukemia (CML) patients and selected individuals of Sri Lanka employing one dimensional sodium dodecyl sulphate poly acrylamide gel electrophoresis (1D-SDS-PAGE) combined with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI–TOF-MS). Erythrocyte membranes from blood were isolated by osmotic lysis, centrifugation and washings. ERMBPs were separated on 1D-SDS-PAGE, visualized by silver staining and the separated protein bands dissected out from the gel and were subjected to digestion by proteolytic enzyme, trypsin, and the resulting peptide mixture was analyzed by MALDI-TOF-MS. Resulting experimental peptide mass values were analyzed by peptide mass fingerprint (PMF) technique. From this analysis 10 ERMBPs including α and β spectrin, ankyrin, band 3, band 4.1, band 4.2, band 7, dematin, actin, 55 KDa erythrocyte membrane protein were identified accurately with their primary structure information. The study was able to provide some evidence for Cathepsin associated cleavage of Band 3 anion transport protein in CML patients reported previously. In addition we were able to detect changes in gel bands between healthy controls and CML patients around the area of 20 kDa in the 1 D-SDS-PAGE. It was identified as nuclear protein Dbf 4 related factor 1 isoform 2. Although erythrocytes are devoid of nuclei, such unexpected nuclear proteins have been identified in previous research. We were successful in identifying several human ERMBPs with available resources. As the identified proteins were known to be related to pathology of some of the hematological diseases, this methodology could be extended to detect the protein changes in erythrocyte membrane protein associated diseases. Therefore, this initial research would at some point lead to discovery of biomarkers to these hematological diseases in Sri Lanka.

Highlights • Rhinella marina is a synanthropic amphibian that offers great possibilities for the study of parasite–host relations. • A complex protein profile, including neuroendocrine proteins indicating intense stress in the liver of the host. • Important aspects of the host’s immune response plasticity are shown. • This study contributes to knowledge of the biochemical aspects of the helminth–host interface., The success of the helminth–host relationship depends on a biochemical molecular arsenal. Perhaps the proteome is the largest and most important set of this weaponry, in which the proteins have a crucial role in vital processes to the parasite/host relationship, from basic metabolism and energy production to complex immune responses. Nowadays, the bioproducts expressed by the parasites are under the “spotlight” of immunoassays and biochemical analysis in helminthology, especially in proteomic analysis, which has provided valuable information about the physiology of the infecting agent. Looking into this point of view, why not turn to the infected agent as well? This study characterised the proteomic profile of fluid-filled fibrous cysts of encapsulated Ortleppascaris sp. larvae in the hepatic parenchyma of their intermediate host, the amphibian Rhinella marina. The proteins were separated by two-dimensional electrophoresis and identified by MS with the aid of Peptide Mass Fingerprint. A total of 54 molecules were analysed in this system, revealing a complex protein profile with molecules related to basic metabolic processes of the parasite, energy production, oxi-reduction and oxidative stress processes as well as molecules related to the host response. This study contributes to proteomic studies of protein markers of the development, infectivity, virulence and co-existence of helminths and their hosts.

a b s t r a c t Bioproducts separation and purification processes are an important segment of the biotechnical industry. Bromelain is an enzyme which has great commercial value and is of wide interest in the pharmaceutical, alimentary, and textile industries, among others. The goal of this study was to develop a new method for bromelain purification from the stem residues resulting from agricultural processing of the pineapple plant. Bromelain was purified using two liquid chromatography steps, ion exchange plus gel filtration chromatography. Use of the methodology which was developed produced an enzyme with a molecular weight of 30 kDa (confirmed by SDS-PAGE), high recovery of enzymatic activity (89%), and with a purifi- cation factor of 16.93, a result superior to the methodologies described in the literature. HPLC showed the presence of two peaks in the ion exchange chromatogram and only one protein in the gel filtration chromatogram. Results indicate that, depending on the destination of the bromelain, the process can be stopped after the first purification step. The MALDI-TOF MS provided the peptide mass fingerprint of bromelain and MALDI-MS/MS the fragmentation profile and sequencing of the ions of m/z 951 and 1584. Thus, the connectivity and chemical structure of bromelain was confirmed. Moreover, besides its superiority to other methodologies, it can be applied to take advantage of the agricultural and industrial pineapple plant residues.

A protein with bioactive potential was purified from the leaves of Datura inoxia P.mil. The preliminary screening of the crude proteins fractionated by Ammonium Sulphate was carried out for its antibacterial, antioxidant and anticancer properties. It exerted antibacterial activity against three bacteria namely Vibrio cholerae, Staphylococcus aureus, and Escherichia coli but was not effective against Aeromonas hydrophilia. It unveiled an increasing free radical scavenging activity in a dose-dependent manner and presented a potential anticancer activity against human colon adenocarcinoma cell line HT 29. The active protein fraction was purified by ion exchange chromatography on CM-Sephadex. The purified protein has a molecular weight of 14.3 kDa and Peptide Mass Fingerprint using Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectroscopy (MALDI-TOF/MS) revealed peptide matches to defensin-like protein of Brassica napus, chaperonin CPN60-2 and mitochondria of Cucurbita maxima. The purified protein showed anticancer activity against HT 29 cancer cell line.

Determination of differentially expressed protein profile is necessary to understand the host response to viral infection. Proteomics can be applied as a tool to examine white shrimp Litopenaeus vannamei molecular responses against white spot syndrome virus (WSSV) infection, thus enabling development of effective strategies to reduce their impact on farms. In the present study, specific pathogen-free shrimp was tested against WSSV infection under several time intervals. Shrimps were submitted to a viral load of with 5.5 9 10 6 viral copies in 100 lL/shrimp. The monitoring of infection was performed in intervals of 6, 12, 24, 48 and 72 h after infection. The analysis was realized using 2-DE, and differentially expressed proteins were identified by MALDI-TOF mass spectrometry (MS) peptide mass fingerprint (PMF). Between the differentially expressed proteins found in the infected animals, the most important were identified as caspase-2, ubiquitin and F1-ATP synthase. They are interesting candidates for biomarkers because could be related to the beginning of apoptosis process. The differentially expressed protein profile creates a new paradigm in the analysis of L. vannamei shrimp molecular response to WSSV infection and in virus-host relationship. Furthermore, it proposes potential bio- markers that allow strategies both selecting less susceptible individuals and reducing the impact of viruses on farms.

The protein profile of latex of Euphorbia nivulia Buch.-Ham. is established. Three new proteins viz., Nivulian-I, II and III have been purified to homogeneity from the latex. The relative molecular masses of Nivulian-I, II and III are 31,486.985, 43,670.846 and 52,803.470 Da respectively. Nivulian-I is a simple type of protein while Nivulian-II and III are glycoproteins. Peptide mass fingerprint analysis revealed peptides of these proteins match with Tubulin alpha-1 chain of Eleusine indica, Maturase K of Banksia quercifolia and hypothetical protein of Zea mays respectively. Tryptic digestion profile of Nivulian-I, II and III, infer the exclusive nature of latex origin proteins and may be new and are additive molecules in the dictionaries of phytoproteins or botany. This is the first of its kind, regarding characterization and validation of Nivulian-I, II and III with respect to peptide sequencing.