Matrix-assisted Laser Desorption/ionization Tandem Time-of-flight Mass Spectrometry Research Articles

Metabolomic approaches for the determination of metabolites from pathogenic microorganisms: A review

Metabolomics is a high precision analytical approach to obtaining detailed information of varieties of metabolites produced in biological systems, including foods. This study reviews the use of metabolomic approaches such as liquid chromatography mass spectrometry (LCMS), gas chromatography mass spectrometry (GC-MS), matrix assisted laser desorption /ionization tandem time of flight mass spectrometry (MALDI-TOF-MS) and nuclear magnetic resonance (NMR) for investigating the presence of foodborne pathogens and their metabolites. Pathogenic fungi and their notable metabolites (mycotoxins) have been studied more extensively using metabolomics as compared to bacteria, necessitating further studies in this regard. Nevertheless, such identified fungal and bacteria metabolites could be used as biomarkers for a more rapid detection of these pathogens in food. Other important compounds detected through metabolomics could also be correlated to functionality of these pathogenic strains, determined by the composition of the foods in which they exist, thereby providing insights into their metabolism. Considering the prevalence of these food pathogens, metabolomics still has potentials in the determination of food-borne pathogenic microorganisms especially for the determination of pathogenic bacteria toxins and is expected to generate research interests for further studies and applications.

Profiling of schizophrenia-associated serum peptides by MALDI-TOF-MS.

Schizophrenia is a psychiatric condition characterized by poor prognosis and severe symptoms that decrease the quality of life of patients. It is therefore important to develop rapid and reliable methods for early diagnosis. To achieve this aim, identification of accurate biomarkers will promote research into the mechanisms of schizophrenia and the design of effective diagnosis tools. Discriminative peptides in the serum of participants (277 schizophrenia patients and 294 healthy controls) were detected using the matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS). The diagnostic model for schizophrenia was validated using the ClinProTool software. Discrimination models were tested in the training set (200 schizophrenia patients and 200 healthy controls), and the robustness of the models was evaluated using the independent test set (77 cases and 94 controls). A total of 77 peaks were significantly different between schizophrenia patients and healthy controls with a signal-to-noise ratio of over 5. Among them, 35 peptides were down-regulated and 42 peptides were up-regulated in schizophrenia patients. With a cross-validation rate of 92.7% and a recognition capability rate of 96.5%, the supervised neural network comprising 25 discriminative peaks was found to be the most efficient model for schizophrenia diagnosis (sensitivity = 96.10%, specificity = 94.68%). Peptides at m/z = 2022.18 corresponded to complement C3f, and peptides at m/z = 1020.89, 1351.02, 1466.1 were identified as fragments of fibrinopeptide A. These two peptides may be potential biomarkers for schizophrenia in the Chinese population. The serum peptide levels present a potential clinical diagnostic tool for schizophrenia.

Microbial assisted (Bacillus mojavensis) production of bio-surfactant lipopeptide with potential pharmaceutical applications and its characterization by MALDI-TOF-MS analysis

Lipopeptides are a fascinating group of biosurfactants possess remarkable surface properties. Functional groups on the surface of these molecules can combine with other substances leading to obtain applicable materials. The aim of the present study is the isolation of an anionic lipopeptide- producing bacterium and verifying its potential for pharmaceutical applications. A Bacillus mojavensis strain capable of producing a great amount of lipopeptide biosurfactant was selected from a number of isolated Bacillus using oil spreading technique, blood agar hemolysis, drop collapsing assay, pH sensitivity and biuret test. Anionic extracellular lipopeptides compound was extracted using metal salt precipitation method and its properties were investigated by TLC, FTIR and SDS page. HPLC-amino acid analysis showed that negative- charge amino acids were dominant in the peptidyl part of the crude supernatant and purified anionic fraction. Furthermore Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS) analysis was applied to detect the lipopeptides harvested from the most anionic fraction obtained from anion- exchange chromatography and the results revealed the presence of surfactin isoform in the extracted substance. The extracted lipopeptides showed surface tension reducing ability, antibacterial activity and lack of toxicity to PC12 and PBMC cells. Also with negative nature interacted with basic molecules to make new compounds, so these surfactants can be considered as a natural product with potential pharmaceutical applications.

Identification of a novel cofilin-related molecule (Der f 31) as an allergen from Dermatophagoides farinae

House dust mite (HDM) allergen is a major cause of allergic disease. In this study, two-dimensional immunoblot and Matrix-Assisted Laser Desorption Ionization tandem Time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify Der f 31. After Der f 31 was cloned, expressed and purified, skin prick test (SPT), Immune inhibitory assays, Western blot, ELISA and asthmatic mouse model were employed to examine the allergenicity of recombinant Der f 31. The gene of Der f 31 includes 447 bps, and encoded 148 amino acids. Positive responses of SPT to r-Der f 31 were 32.5% in 43 HDM-allergic patients. r-Der f 31 can induce allergic pulmonary inflammation in the mouse model. In conclusion, Der f 31 is a novel subtype of dust mite allergens.

Proteomic identification of predictive tissue biomarkers of sensitive to neoadjuvant chemotherapy in squamous cervical cancer

AimsThe regimens of neoadjuvant chemotherapy (NAC) in squamous cervical cancer (SCC) frequently use cisplatin combined with paclitaxel. Unfortunately, some cervical cancers show resistance to the principal chemotherapeutic agents in the treatment, decreasing the effectiveness of this therapy. The objective of this study was to search for predictive markers of response to NAC in patients with SCC. Main methodsTwo-dimensional gel electrophoresis (2-DE) accompanied by matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS) was used to analyze and identify differentially expressed proteins in ten cases of advanced cervical cancer patients receiving cisplatin-based NAC. Each of these patients received more than two cycles of NAC. Cell proliferation rate in cisplatin resistant human cervical cancer cell Hela/DDP and its parent cell Hela after treatment with Hsp70 inhibitor and/or cisplatin were tested by MTT assay. Key findingsTwelve protein spots changed in abundance, quantitative comparison of spot volumes showed that seven protein spots were up-regulated and five spots were down-regulated in the NAC non-responders compared to the NAC responders. These proteins are involved in various cellular processes essential for cell metabolism, migration and apoptotic signal transduction. The high-fold changes proteins of stathmin1, Hsp70 and pyruvate kinase isoform M2 were validated by Western blot analysis. Over-expression of Hsp70 inhibits the efficacy of cisplatin. Hsp70 inhibitor enhanced the sensitivity to cisplatin in both Hela and Hela/DDP cells. SignificanceThe study found many candidate proteins involved in chemotherapy resistance/sensitivity, among them Hsp70 might be potential biomarkers to predict the efficacy of chemotherapy for SCC patients.

Differential Proteomics Analysis of Colonic Tissues in Patients of Slow Transit Constipation.

Objective. To investigate and screen the different expression of proteins in STC and normal group with a comparative proteomic approach. Methods. Two-dimensional electrophoresis was applied to separate the proteins in specimens from both 5 STC patients and 5 normal controls. The proteins with statistically significant differential expression between two groups were identified by computer aided image analysis and matrix assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI-TOF-MS). Results. A total of 239 protein spots were identified in the average gel of the normal control and 215 in patients with STC. A total of 197 protein spots were matched and the mean matching rate was 82%. There were 14 protein spots which were expressed with statistically significant differences from others. Of those 14 protein spots, the expression of 12 spots increased markedly, while that of 2 spots decreased significantly. Conclusion. The proteomics expression in colonic specimens of STC patients is statistically significantly different from that of normal control, which may be associated with the pathogenesis of STC.

Discovery of novel protein biomarkers in early silicosis by proteomics and identification of alpha B-crystallin

To establish 2-dimensional gel electrophoresis (2-DE) image for the early lung injury rats induced by silica dioxide and to identify differentially expressed protein biomarkers during the early stage of silicosis. The animal models of silicosis were established and morphology changes were observed by HE staining, and then the key protein biomarkers in silicosis were identified by 2-DE and matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI-TOF-MS) and verified by Western blot. The well qualified 2-DE gel images for experimental and control lung tissues were successfully established, and 40 different protein spots was observed when comparing the gel images between the experimental and control groups. Twenty of them were analyzed by MALDI-TOF-MS. A total of 13 altered proteins were identified, including alpha B-crystallin, mast cell protease 6, annexin 1, etc. The expression of alpha B-crystallin in lung was further verified by Western blot. The protein expression of alpha B-crystallin was increased significantly, suggesting that it might play an important role in the progress of silicosis.

Effects of anthracene on filtration rates, antioxidant defense system, and redox proteomics in the Mediterranean clam Ruditapes decussatus (Mollusca: Bivalvia).

This study aimed at analyzing the impact of a toxic polyaromatic hydrocarbon (PAH), anthracene (ANT), on Ruditapes decussatus collected from a Tunisian coastal lagoon (Bizerte Lagoon). Filtration rates, several antioxidant enzymes--superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione transferase (GST)--as well as indices of protein oxidation status were determined in various tissues of this bivalve. Specimens were exposed to 100 μg/L of ANT for 2 days. ANT levels were evaluated using HPLC and were detected in the gill and digestive gland at different amounts. ANT exposure altered the behavior of bivalves by changing the siphon movement and decreasing filtration rate significantly. The enzymatic results indicated that ANT exposure affected the oxidative stress status of the gills of R. decussatus. In addition, modification of proteins was detected in the gills using redox proteomics after ANT treatment. Three protein spots were successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-MS). These proteins can be roughly related to muscle contraction function. In contrast, no significant modification of enzymatic and protein responses was detected in the digestive gland after ANT treatment. These data demonstrate that combined behavioral and biochemical analyses are a powerful tool to provide valuable insights into possible mechanisms of toxicity of anthracene in R. decussatus. Additionally, the results highlight the potential of the gill as a valuable candidate for investigating PAH toxicity.

Graphene/TiO2 nanocomposite based solid-phase extraction and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for lipidomic profiling of avocado (Persea americana Mill.)

Phospholipids possess important physiological, structural and nutritional functions in biological systems. This study described a solid-phase extraction (SPE) method, employing graphene and titanium dioxide (G/TiO2) nanocomposite as sorbent, for the selective isolation and enrichment of phospholipids from avocado (Persea americana Mill.). Based on the principal that the phosphoryl group in the phospholipid can interact with TiO2 via a bridging bidentate mode, an optimum condition was established for SPE, and was successfully applied to prepare avocado samples. The extracts were monitored by matrix-assisted laser desorption ionization time-of-flight/tandem mass spectrometry (MALDI-TOF/MS) in both positive-ion and negative-ion modes. Results showed that phospholipids could be efficiently extracted in a clean manner by G/TiO2 based SPE. In addition, the signals of phospholipids were enhanced while the noise was reduced. Some minor peaks became more obvious. In conclusion, the nanocomposite material of G/TiO2 was proved to be a promising sorbent for selective separation of phospholipids from crude lipid extract.

Study on differentially expressed serum proteins in Uyghur patients with primary hyperuricemia

2-Dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS) were used to screen differentially expressed serum proteins in Uyghur patients with hyperuricemia and control subjects.Western blot was used to validate differential proteins.Gel-image analysis revealed that there were 11 differential protein spots.A total of 4 differential protein spots were successfully identified by MALDI-TOF-MS.Complement 3,complement 4,haptoglobin,and apolipoprotein Al levels were up-regulated in patients as compared with controls.The result of complement 3 expression was validated by Western blot,which is accordant with that of proteomics. Key words: Hyperuricemia ; Uyghur; Serum ; Proteomics

Differential protein expression between EBV-positive and negative epithelial cells

Epstein Barr virus infection is believed to play a role in the development of nasopharyngeal carcinoma. In order to investigate the function of EBV in epithelial cell, proteomic methods were used to find and identify the differential proteins and expected to elucidate the mechanism of EBV. Altered protein expressions were found between 293 cell (HEK293) and EBV infected cell (293-EBV). In this study, we separated differential expressed proteins using 2D-DIGE method while matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry (MALDI-TOF-MS) method was used to identify proteins. The results showed that 14 proteins were up regulated and 3 proteins were down regulated in 293-EBV cells. Bioinformatic analysis showed that these proteins are involved in cell proliferation, metastasis, apoptosis, metabolism, and signal transduction. Western blotting analysis was further carried out to verify the MS results. Thus, EBV may exert its functions by mediating differential expression of these proteins.

Protein Profile of Bacillus subtilis Spore

Natural wild-type strains of Bacillus subtilis spore is regarded as a non-pathogenic for both human and animal, and has been classified as a novel food which is currently being used as probiotics added in the consumption. To identify B. subtilis spore proteins, we have accomplished a preliminary proteomic analysis of B. subtilis spore, with a combination of two-dimensional electrophoretic separations and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI-TOF-MS). In this article, we presented a reference map of 158 B. subtilis spore proteins with an isoelectric point (pI) between 4 and 7. Followed by mass spectrometry (MS) analysis, we identified 71 B. subtilis spore proteins with high level of confidence. Database searches, combined with hydropathy analysis and GO analysis revealed that most of the B. subtilis spore proteins were hydrophilic proteins related to catalytic function. These results should accelerate efforts to understand the resistance of spore to harsh conditions.

Haemocyte protein expression profiling of scallop Chlamys farreri response to acute viral necrosis virus (AVNV) infection

Acute viral necrosis virus (AVNV) was newly reported as one causative agent responsible for mass mortality of adult Chinese scallop Chlamys farreri, which is widely cultured on northern China coast. Unfortunately, the interaction between virus and host is largely unknown. According to these, this study was undertaken to deeply explore the immune response of haemocyte against AVNV. Two-dimensional gel electrophoresis (2-DE) was introduced to produce protein expression profiles from samples taken at 24 h post-infection (hpi) from the haemocytes of C. farreri that were either specific pathogen free or else infected with AVNV. Forty-eight protein spots, which consistently showed either a marked change (≥1.5-fold difference) in accumulated levels or else were highly expressed in haemocytes, were selected for further investigation. In-gel trypsin digestion was conducted followed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-MS). Matching search was subsequently performed throughout bioinformatics databases. A total of 42 proteins were identified, all of which were classified into eight categories according to their Gene Ontology annotations of biological processes and molecular functions, i.e. cytoskeleton proteins, proteins involved in metabolism, proteins related to calcium homeostasis, chaperone, proteins involved in immunity, proteins involved in transcriptional regulation, proteins related to signal transduction, and ungrouped proteins. The possible biological significance of some observed proteins in the host response to AVNV was discussed. These studies could be served as the first global analysis of differentially expressed proteins in haemocytes from AVNV-infected C. farreri, and in addition to increasing our understanding of the pathogenesis of this virus-associated scallop disease, the results presented here should be useful both for potential biomarkers identification and anti-virus approaches development as well.

Affinity labeling coupled with matrix assistant laser desorption tandem time of flight mass spectrometry for quantitative proteomics research

A relative quantitative method for differential proteomics by cleavable isotope-coded affinity tag (cICAT) and matrix-assisted laser desorption/ionization tandem timeof-flight mass spectrometry (MALDI-TOF-MS) was established. The accuracy and reproducibility of the method were evaluated by bovine serum albumin (BSA) digest as having a relative standard deviation of less than 30% and good reproducibility. The dynamic range was also evaluated by analyzing two mixtures of several standard proteins with different concentration. The experimental results showed that in the dynamic range of 1:30, the quantitation error of the method was less than 30%. Although the quantitation error becomes very large when used beyond this range, it does not affect the derivation of information on the differential proteins. All the work provides an alternative method for differential proteomics analysis in biological samples from different origins.

Identification of covalent modifications in P450 2E1 by 1,2-epoxy-3-butene in vitro

1,3-Butadiene is metabolized mainly by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3-butene (EB), a reactive metabolite. It has been shown that P450s can be inactivated by covalent binding of reactive metabolites to protein or heme. Molecular dosimetry studies have clearly shown that BD metabolism follows a supralinear dose response, suggestive of saturation of metabolic activation. In this study, potential binding sites of EB in human P450 2E1 were identified and modeled to test whether EB covalently binds to residues important for enzyme activity. Commercially available human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization tandem Time-of-Flight mass spectrometry (MALDI-MS). The identity of EB modified peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization tandem mass spectrometry (MALDI-MS/MS) sequencing. It was shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of eight peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His(109), His(370)) are clearly associated with the active site, and that their Calpha atoms are located less than 9A from a known inhibitor binding site. In addition, the side chain of His(370) is within 4A of the heme group and its modification is expected to influence the orientation of the heme. The Calpha atom of Tyr(71) is within 14A of the potential inhibitor binding site and within 7A of the flap undergoing conformational change upon ligand binding, potentially placing Tyr(71) near the substrate as it enters and leaves the active site. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory mechanism for BD metabolism.