| 1. |
- Bllaci, Loreta, et al.
(author)
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Fast Surface Acoustic Wave-Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Cell Response from Islets of Langerhans.
- 2013
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 85:5, s. 2623-2629
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Journal article (peer-reviewed)abstract
- A desire for higher speed and performance in molecular profiling analysis at a reduced cost is driving a trend in miniaturization and simplification of procedures. Here we report the use of a surface acoustic wave (SAW) atomizer for fast sample handling in matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) peptide and protein profiling of Islets of Langerhans, for future type 2 diabetes (T2D) studies. Here the SAW atomizer was used for ultrasound (acoustic) extraction of insulin and other peptide hormones released from freshly prepared islets, stimulated directly on a membrane. A high energy propagating SAW atomizes the membrane-bound liquid into approximately 2 μm diameter droplets, rich in cell-released molecules. Besides acting as a sample carrier, the membrane provides a purification step by entrapping cell clusters and other impurities within its fibers. A new SAW-based sample-matrix deposition method for MALDI MS was developed and characterized by a strong insulin signal, and a limit of detection (LOD) lower than 100 amol was achieved. Our results support previous work reporting the SAW atomizer as a fast and inexpensive tool for ultrasound, membrane-based sample extraction. When interfaced with MALDI MS, the SAW atomizer constitutes a valuable tool for rapid cell studies. Other biomedical applications of SAW-MALDI MS are currently being developed, aiming at fast profiling of biofluids. The membrane sampling is a simplistic and noninvasive collection method of limited volume biofluids such as the gingival fluid and the tearfilm.
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| 2. |
- Don-Doncow, Nicholas, et al.
(author)
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Galiellalactone is a Direct Inhibitor of STAT3 in Prostate Cancer Cells.
- 2014
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In: Journal of Biological Chemistry. - 1083-351X. ; 289:23, s. 15969-15978
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Journal article (peer-reviewed)abstract
- The transcription factor STAT3 is constitutively active in several malignancies including castration-resistant prostate cancer and has been identified as a promising therapeutic target. The fungal metabolite galiellalactone, a STAT3 signaling inhibitor, inhibits the growth, both in vitro and in vivo, of prostate cancer cells expressing active STAT3 and induces apoptosis of prostate cancer stem cell-like cells expressing pSTAT3. However, the molecular mechanism of this STAT3 inhibiting effect by galiellalactone has not been clarified. A biotinylated analogue of galiellalactone (GL-biot) was synthesized to be used for identification of galiellalactone target proteins. By adding streptavidin-sepharose beads to GL-biot treated DU145 cell lysates, STAT3 was isolated and identified as a target protein. Confocal microscopy revealed GL-biot in both the cytoplasm and nucleus of DU145 cells treated with GL-biot, appearing to co-localize with STAT3 in the nucleus. Galiellalactone inhibited STAT3 binding to DNA in DU145 cell lysates without affecting phosphorylation status of STAT3. Mass spectrometry analysis of recombinant STAT3 protein pretreated with galiellalactone revealed three modified cysteines (cys-367, cys-468 and cys-542). We here demonstrate with chemical and molecular pharmacological methods that galiellalactone is a cysteine reactive inhibitor that covalently binds to one or more cysteines in STAT3 and that this leads to inhibition of STAT3 binding to DNA and thus blocks STAT3 signaling without affecting phosphorylation. This further validates galiellalactone as a promising direct STAT3 inhibitor for treatment of castration-resistant prostate cancer.
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| 3. |
- Jovic, Sandra, et al.
(author)
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Expression of MIG/CXCL9 in Cystic Fibrosis and Modulation of Its Activities by Elastase of Pseudomonas aeruginosa.
- 2014
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In: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 6:6, s. 846-859
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Journal article (peer-reviewed)abstract
- In cystic fibrosis (CF), colonization of the airways with Pseudomonas aeruginosa is associated with disease deterioration. The mechanism behind the disease progression is not fully understood. The present work shows that the antibacterial chemokine MIG/CXCL9 is present in the airways and in sputum of CF patients. MIG/CXCL9 showed high bactericidal activity against. P. aeruginosa, including some strains from the airways of CF patients. Full-length MIG/CXCL9 was detected in sputum from healthy controls and CF patients colonized with P. aeruginosa. However, degraded MIG/CXCL9 was only found in CF sputum. In vitro, elastase of P. aeruginosa cleaved off a fragment of similar size and two additional fragments from MIG/CXCL9. The fragments showed less bactericidal activity against P. aeruginosa compared with the full-length protein. The fragments did not activate the MIG/CXCL9 receptor CXCR3 (expressed e.g. by NK cells, mast cells, and activated T cells) but instead displayed noncompetitive inhibition. In vitro, a decrease in CXCR3-bearing cells was found within and in the proximity of the bronchial epithelium of CF lung tissue compared with controls. Taken together, both bactericidal and cell-recruiting activities of MIG/CXCL9 are corrupted by P. aeruginosa through release of elastase, and this may contribute to impaired airway host defense in CF. © 2014 S. Karger AG, Basel.
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| 4. |
- Lambert, Wietske, et al.
(author)
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Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry.
- 2012
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In: Cell Stress & Chaperones. - : Springer Science and Business Media LLC. - 1466-1268 .- 1355-8145.
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Journal article (peer-reviewed)abstract
- Small heat-shock protein chaperones are important players in the protein quality control system of the cell, because they can immediately respond to partially unfolded proteins, thereby protecting the cell from harmful aggregates. The small heat-shock proteins can form large polydisperse oligomers that are exceptionally dynamic, which is implicated in their function of protecting substrate proteins from aggregation. Yet the mechanism of substrate recognition remains poorly understood, and little is known about what parts of the small heat-shock proteins interact with substrates and what parts of a partially unfolded substrate protein interact with the small heat-shock proteins. The transient nature of the interactions that prevent substrate aggregation rationalize probing this interaction by crosslinking mass spectrometry. Here, we used a workflow with lysine-specific crosslinking and offline nano-liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry to explore the interaction between the plant small heat-shock protein Hsp21 and a thermosensitive model substrate protein, malate dehydrogenase. The identified crosslinks point at an interaction between the disordered N-terminal region of Hsp21 and the C-terminal presumably unfolding part of the substrate protein.
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| 5. |
- Lindberg, Claes, et al.
(author)
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Liquid chromatography-tandem mass spectrometry approach for quantification of mucins from sputum using C-13,N-15-labeled peptides as internal standards
- 2013
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In: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 434:1, s. 84-92
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Journal article (peer-reviewed)abstract
- Mucins are of great interest owing their involvement in physiological and pathological processes in the airways. A method which allows accurate quantification of such proteins in sputum samples may be helpful for research in this field. A liquid chromatographic single reaction monitoring (SRM) method was developed for the quantification of two mucins, MUC5AC and MUC5B, in induced sputum samples. Sample preparation for the assay included solubilization, reduction and alkylation prior to tryptic digestion. Solid phase extraction using C(18) sorbent was used for sample clean up prior to the LC-MS/MS analysis. A cysteine containing peptide was selected for quantification of MUC5AC protein, whereas a non-cysteine peptide was used for the quantification of MUC5B protein. Stable isotope-labeled synthetic peptides were used as internal standards and linear calibration curves were constructed in the range of 0.3 - 40 pmol/L. Both mucins could be determined with a precision of 6-19% and an accuracy of 98-114%. The method is transferable to robotics and is suitable to be run in a 96-well format.
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| 6. |
- Miliotis, Tasso, et al.
(author)
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Quantitative high-performance liquid chromatography-tandem mass spectrometry method for the analysis of free desmosines in plasma and urine
- 2013
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In: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1308, s. 73-78
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Journal article (peer-reviewed)abstract
- A rapid method for the determination of the sum of free desmosine and isodesmosine in human plasma and urine is described. Efficient sample clean-up prior to LC-MS/MS analysis is mandatory for detection of free desmosines in plasma samples. The combination of ultra-filtration and a two-step solid phase extraction minimizes the sample complexity and ion suppression effects. The flow through from the ultra filtration is passed through a C18 resin and then the target analytes are trapped and enriched on a mixed mode solid phase extraction material. The combination of these three orthogonal sample preparation steps allows detection of endogenous free desmosines in plasma from healthy individuals. An analytical column packed with porous graphitic carbon material enables the retention of the polar desmosine analytes, which are measured by electrospray ionization tandem mass spectrometry. Deuterium labeled isodesmosine is added as internal standard and a linear calibration curve was constructed in the range of 0.1-2.0 nmol/L for plasma samples and 5-200 nmol/L for urine samples. These results demonstrate that the described LC-MS/MS method provides sensitive, repeatable and accurate quantification of free desmosines in plasma and urine samples. (C) 2013 Elsevier B.V. All rights reserved.
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| 7. |
- Söderberg, Christopher, et al.
(author)
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Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation.
- 2012
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:6
-
Journal article (peer-reviewed)abstract
- Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using (14)N/(15)N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers.
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| 8. |
- Yakovleva, Maria, et al.
(author)
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Further Insights into the Catalytical Properties of Deglycosylated Pyranose Dehydrogenase from Agaricus meleagris Recombinantly Expressed in Pichia pastoris
- 2013
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 85:20, s. 9852-9858
-
Journal article (peer-reviewed)abstract
- The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris. Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 degrees C. The remaining larger fragment has a molecular weight of similar to 46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, "wired" to graphite electrodes with two different osmium redox polymers: [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(poly(vinylimidazole))(10)Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)(2)(poly-(vinylimidazole))(10)Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with J(max). 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N-75, N-175, and N-252 were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.
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