Evolution of proteomics technologies for understanding respiratory syncytial virus pathogenesis.

Introduction: Respiratory syncytial virus (RSV) is a major human pathogen associated with long term morbidity. RSV replication occurs primarily in the epithelium, producing a complex cellular response associated with acute inflammation and long-lived changes in pulmonary function and allergic disease. Proteomics approaches provide important insights into post-transcriptional regulatory processes including alterations in cellular complexes regulating the coordinated innate response and epigenome.Areas covered: Peer-reviewed proteomics studies of host responses to RSV infections and proteomics techniques were analyzed. Methodologies identified include 1)." bottom-up" discovery proteomics, 2). Organellar proteomics by LC-gel fractionation; 3). Dynamic changes in protein interaction networks by LC-MS; and 4). selective reaction monitoring MS. We introduce recent developments in single-cell proteomics, top-down mass spectrometry, and photo-cleavable surfactant chemistries that will have impact on understanding how RSV induces extracellular matrix (ECM) composition and airway remodeling.Expert opinion: RSV replication induces global changes in the cellular proteome, dynamic shifts in nuclear proteins, and remodeling of epigenetic regulatory complexes linked to the innate response. Pathways discovered by proteomics technologies have led to deeper mechanistic understanding of the roles of heat shock proteins, redox response, transcriptional elongation complex remodeling and ECM secretion remodeling in host responses to RSV infections and pathological sequelae.

Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris.

Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man9GlcNAc2- to Man15GlcNAc2- with major Man10GlcNAc2- and Man11GlcNAc2- species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE- or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction.

Interferon γ induced compositional changes in human bone marrow derived mesenchymal stem/stromal cells.

BACKGROUND: Mesenchymal stem/stromal cells (MSC) display a range of immunoregulatory properties which can be enhanced by the exposure to cytokines such interferon γ (IFN-γ). However the compositional changes associated with the 'licensing' of these cells have not been clearly defined. The present study was undertaken to provide a detailed comparative proteomic analysis of the compositional changes that occur in human bone marrow derived MSC following 20 h treatment with IFN-γ. METHODS: 2D LC MSMS analysis of control and IFN-γ treated cells from 5 different healthy donors provided confident identification of more than 8400 proteins. RESULTS: In total 210 proteins were shown to be significantly altered in their expression levels (≥|2SD|) following IFN-γ treatment. The changes for several of these proteins were confirmed by flow cytometry. STRING analysis determined that approximately 30% of the altered proteins physically interacted in described interferon mediated processes. Comparison of the list of proteins that were identified as changed in the proteomic analysis with data for the same proteins in the Interferome DB indicated that ~35% of these proteins have not been reported to be IFN-γ responsive in a range of cell types. CONCLUSIONS: This data provides an in depth analysis of the proteome of basal and IFN-γ treated human mesenchymal stem cells and it identifies a number of novel proteins that may contribute to the immunoregulatory capacity if IFN-γ licensed cells.

Salivary Proteomic Analysis of Canine Oral Melanoma by MALDI-TOF Mass Spectrometry and LC-Mass Spectrometry/Mass Spectrometry.

Canine oral melanoma (COM) is a common oral cancer with aggressiveness and high metastasis. A tumor in a dog's mouth makes it difficult to be observed at the early-clinical stage. Salivary biomarkers may be useful for early detection, prognosis, and monitoring of therapies. In addition, salivary collection is a simple and non-invasive technique. The present study describes how to identify salivary biomarkers in COM using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) approaches. Western blot analysis has been used to confirm the protein expression. The sequence of expressed protein from western blot has been verified by LC-MS/MS.

Potential Use of Vacuum Impregnation and High-Pressure Homogenization to Obtain Functional Products from Lulo Fruit (Solanum quitoense Lam.).

Lulo (Solanum quitoense Lam.) is a Colombian fruit that is mostly used in the preparation of homemade juice as well as natural remedy for hypertension. The aim of this study was to determine physicochemical and antioxidant properties (antioxidant capacity, total phenols, flavonoids and spermidine content, and polyphenolic compounds profile by liquid chromatography-mass spectrometry (LC-MS)) of the lulo fruit and its juice. Additionally, vacuum impregnation (VI) properties of the fruit and the effect of high homogenization pressure (50, 100, and 150 MPa) on the juice properties were studied. The results revealed a good availability and impregnation capacity of the pores in fruits with similar maturity index. The main differences observed between the juice and fruit derive from removing solids and bioactive components in the filtering operation. However, the effect of high-pressure homogenization (HPH) on particle size and bioactive compounds increases the antiradical capacity of the juice and the diversity in polyphenolics when increasing the homogenization pressure.

Compositional Characteristics of Glucuronide Conjugates in Regional Glechoma hederacea var. longituba Herbal Extracts Using a Set of Polyphenolic Marker Compounds.

Glechoma hederacea var. longituba (GHL) is one of many herbal plants widely used in hot herbal teas and in oriental prescriptions to treat various diseases. Although the beneficial effects of GHL may be influenced by differences in the composition of active constituents in the herbal extracts, there are few reports on the compositional characteristics of GHL herbal extracts to date. In this study, liquid chromatography-mass spectrometry technology was used for comparative analysis of constituents in hot-water extracts of GHL samples obtained from various cultivating provinces in South Korea. A set of marker panel consisting of nine polyphenolic compounds, including glucuronide conjugates in particular, was constructed and used to monitor the compositional characteristics in each GHL extract. Our findings show that some of the marker compounds, including rosmarinic acid, were persistently observed as major constituents in the analyses of the 22 GHL sample extracts, whereas, interestingly, other marker compounds such as polyphenol-glucuronic acid conjugates displayed dramatic differences in compositional ratios. This chromatographic approach using the marker compound panel can be applied to qualitatively and quantitatively evaluate compositional characteristics in the GHL extracts, and can also be useful for quality assays of the GHL herbal plant in medicinal and industrial fields.

Flavonoids and caffeoylquinic acids in Chrysanthemum morifolium Ramat flowers: A potentially rich source of bioactive compounds.

Varieties of chrysanthemums are among the world's most valuable edible ornamental crops. However, the availability and relationship between the bio-chemicals of chrysanthemums and their morphological variations remain unclear. We developed liquid chromatography mass spectrometry to construct a spectral tag library to identify and quantify chemicals of 7 caffeoylquinic acids, 21 flavones and flavonols, 4 carotenoids, and 13 other compounds in 27 cultivars and representative tea of Chrysanthemum morifolium. A correlation analysis found that more acacetin 7-O-galactoside (23) resulted in lighter colored flowers and less acacetin (43) and kaempferol (44) was associated with yellow flowers. Hot-H2O extraction of C. morifolium tea showed that most flavonoids and caffeoylquinic acids dissolved out at 30 min, with 20.977 and 8.958 mg/g GW indicated that C. morifolium, which is used in food and tea, is rich in flavonoids and carotenoids. The results improve our understanding of flavonoid biosynthesis and the mechanisms responsible for flower color.

Recent Progress in FD-LC-MS/MS Proteomics Method.

Through the course of our bio-analytical chemistry studies, we developed a novel proteomics analysis method, FD-LC-MS/MS (fluorogenic derivatization-liquid chromatography-tandem mass spectrometry). This method consists of fluorogenic derivatization (FD), LC separation, and detection/quantification of the derivatized proteins, followed by isolation, tryptic digestion of the isolated proteins, and final identification of the isolated proteins using electrospray ionization nano-LC-MS/MS of the generated peptide mixtures with a probability-based protein identification algorithm. In this review, we will present various examples where this method has been used successfully to identify expressed proteins in individual human cells. FD-LC-MS/MS is also suitable for differential proteomics analysis. Here, two biological samples are treated by the same steps mentioned above, and the two chromatograms obtained are compared to identify peaks with different intensities (variation in protein levels). Associated peak fractions are then isolated, and the differentially expressed proteins between the two samples are identified by LC-MS/MS. Several biomarkers for cancers have been identified by FD-LC-MS/MS. For more efficient separation, nano-flow LC with a phenyl-bonded monolithic silica-based capillary column was adopted for cell-expressed intact protein analysis. The derivatized human cell proteins (K562) and yeast cell (Saccharomyces cerevisiae) proteins as model intact cell proteins were analyzed by nano-flow LC with fluorescence detection. More than 1,300 protein peaks were separated/detected from both cells. For straightforward comparison of multiple peak separation profiles, a novel type of chromatogram display, termed the "spiderweb" chromatogram, was developed. A nano-LC-FD-LC-mass spectrometry trial for molecular weight estimation of FD proteins has also been conducted.

Identification of Proteolytic Cleavage Sites of EphA2 by Membrane Type 1 Matrix Metalloproteinase on the Surface of Cancer Cells.

Proteolytic cleavage of membrane proteins can alter their functions depending on the cleavage sites. We recently demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP ) converts the tumor suppressor EphA2 into an oncogenic signal transducer through EphA2 cleavage. The cleaved EphA2 fragment that remains at the cell surface may be a better target for cancer therapy than intact EphA2. To analyze the cleavage site(s) of EphA2, we purified the fragments from tumor cells expressing MT1-MMP and Myc- and 6× His-tagged EphA2 by two-step affinity purification . The purified fragment was digested with trypsin to generate proteolytic peptides , and the amino acid sequences of these peptides were determined by nano-LC-mass spectrometry to identify the MT1-MMP-mediated cleavage site(s) of EphA2.

Comparative Proteomic Profiling of Leishmania tropica: Investigation of a Case Infected with Simultaneous Cutaneous and Viscerotropic Leishmaniasis by 2-Dimentional Electrophoresis and Mass Spectrometry.

BACKGROUND: Viscerotropic leishmaniasis caused by Leishmania tropica poses a significant problem in the diagnosis and treatment management. Since differential gene expression is more important in outcome of the infection, we employed proteomic approach to identify potential proteins involved in visceralization of L. tropica. METHODS: The proteomes profiling of L. tropica isolated from cutaneous and visceral tissues of one host were compared by 2-DE/MS proteomics study. Moreover, the transcript level of some identified proteins was confirmed using real-time RT-PCR. RESULTS: Of the 700 protein spots that were detected reproducibly on each gel, 135 were found to be differentially expressed (P≤ 0.05). Most of responsive proteins in visceral isolate changed in less abundant compared to cutaneous isolate. Among differentially expressed proteins, 56 proteins were confidently identified and classified according to the biological process. The largest groups consist of proteins involved in carbohydrate metabolism and protein synthesis. Most of the identified proteins, which implicated in energy metabolism, cell signaling and virulence were down-regulated, whereas some proteins that have a role in protein folding, antioxidant defense and proteolysis were up-regulated in visceral form. Moreover, the transcript level of some identified proteins such as co-chaperon was confirmed using real-time RT-PCR. CONCLUSION: L. tropica probably uses different mechanisms for survival and multiplication in viscera to establish viscerotropic leishmaniasis. The current study provides some clues into the mechanisms underlying the dissemination of L. tropica .

Determination of carbonyl compounds in particulate matter PM2.5 by in-tube solid-phase microextraction coupled to capillary liquid chromatography/mass spectrometry.

In this paper, a new procedure based on in-tube solid phase microextraction (IT-SPME)-capillary liquid chromatography hyphenated to mass spectrometry detection by using microelectrospray ionisation (CapLC-MS), has been reported. The device was proposed to quantify 12 carbonyl compounds (10 aliphatic aldehydes, an unsaturated aldehyde and a ketone) derivatized with 2,4-dinitrophenylhidrazine (DNPH) reagent in aqueous extracts of PM2.5. This methodology involves the on-line preconcentration of DNPH-carbonyl compounds derivatives coupled to the CapLC-MS system, efficiently providing appropriate sensitivity for the determination of the target analytes. Detection limits for the analytes ranged between 0.9 and 8.2 ng L(-1). These values represent a remarkable improvement over the existing methods since PM2.5 analysis can be carried out avoiding off-line preconcentration steps. The procedure is also proved useful for analysing water samples. Under the optimised conditions, IT-SPME-CapLC-MS shows satisfactory recovery values (80-90%) for spiked samples.