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MASS SPECTROMETRY LABORATORY |
| Short description of laboratory activity | TOP |
The systematic analysis of (bio)molecules is performed through the implication of novel and ultrasensitive methods of high resolution for the detection and sequencing of molecular markers. Mass spectrometry is an analytical tool used for measuring the molecular mass of a sample; it can be coupled with high performance chromatography and chip ionization.
| Analysis and characterization techniques used in our laboratory | TOP |
(ESI) HCT ULTRA Mass Spectrometer
Manufacturer: Bruker Daltonics – Bremen, Germany BiVersa NanoMate 400
Manufacturer: Advion BioSciences, Ithaca, USA |
Technical characteristics HCT
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Applications in:
Electrospray Ionization (ESI) is one of the Atmospheric Pressure Ionization (API) techniques and is well-suited to the analysis of polar molecules ranging from less than 100 Da to more than 1,000,000 Da in molecular mass. The increased performances consist in the possibility of multiple fragmentations up to MS11 through the isolation sequencing cycle, using the ion trap and CID, ETD sources. It can perform very sensitive fragmentations by breaking the peptide covalent bound (in the case of glycopeptides), while the saccharides bonds, although weaker, remain intact. These advantages allow us the structural identification of molecules like proteoglycans, glycosaminoglycans, glycopeptides, glycosphingolipids etc. Among other performances, is also the sensitivity of the analysis meaning the small quantity of sample needed for achieving one spectrum, and the versatility of the method demonstrated by the possibility of changing the infusion source very quickly. The infusion sources produced by Bruker Daltonics, are: on-line microelectrospray, off-line nanoelectrospray and on-line nanoelectrospray.
Fully automated chip-based nanoelectrospray is performed in our laboratory on a NanoMate robot incorporating ESI 400 Chip technology (Advion BioSciences, Ithaca, USA) controlled and manipulated by ChipSoft 8.1.1 software operating under Windows system. The advantages of the NanoMate HCT MSn recommend it as a method of choice in modern glycomics, particularly for clinical applications focused on discovery of carbohydrate biomarkers and their fine structure characterization.
Samples characteristics
The samples which can be analyzed by mass spectrometry could be organic or inorganic, volatile or non-volatile mixtures, with the possibility of “de novo” identification of components even if they are in very small quantity. Among the samples we analyze are the following: sialilated glycosphingolipids, proteoglycans, glycosaminoglycans, glycopeptides, proteins etc. the samples must be diluted in volatile solvents in the purpose of electrospray formation. ESI and nanospray ionization are very sensitive analytical techniques but the sensitivity deteriorates with the presence of non-volatile buffers and other additives, which should be avoided as far as possible.
| Rezultate: | TOP |
(-) chip nanoESI HCT MS of fructooligosacharides synthesized from sucrose and raffinose by the Lsc3 protein, by integrating the gene that expresses it in the genome of E. coli. All ions are singly charged. Gal corresponds to galactose, G to glucose and F to fructose. ♦ = [FnGGal-H+]-; ● = [FnGGal-H++H2O]-; ■ = [FnGGal+HCOO-]-; ▲= [FnGGal+ PO43-+2H+]-; * = [FnGGal+PO43-+2H++H2O]-; ○ = [FnGGal-H-+2H2O]-. MS2 marks the ions that are further fragmented by CID MS/MS. |
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(-) nanoESI chip HCT MS1 of a native ganglioside mixture extracted from an anencephalic fetal brain remnant |
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| In the image above is illustrated a fragmentation spectra of 1063.34 ion corresponding to a ganglioside, GT1 (d18:1/18:0), from a 28 gestational week anencephalic fetus. |
| Short description of the technique: |
For the MS analysis, the stock solution is prepared by dissolving the dry material in methanol and stored at -27˚C. The dilution of the stock solution with pure methanol is made for the purpose of obtaining dried samples at concentrations of 5pmol/μL (calculated for an average molecular mass of 2000 g/mol). All the solutions will be centrifuged (SIGMA 2-16 Sartorius, GmbH) for 2 hours at 5000 r.p.s. because otherwise the chip nozzles will be clogged. The samples with high sodium content will be dialyzed for 4 days in 5mM ammonium acetate at 40˚C using “cut-off” 500 Da membranes from Schuell (Darmstadt, Deutschland).
The solutions are liophilized in an SpeedVac Concentrator, SPD 111V-230 from Thermo Electron Corporation, (Asheville, NC, USA), coupled with a pomp PC 2002 Vario cu controler CVC 2000 from Vaccubrand (Wertheim, Germania).
The robot (NanoMate) was coupled to the HCT Ultra mass spectrometer via an in-laboratory made mounting system. For NanoMate interfacing, the conventional Bruker electrospray ion source was detached and all HCT instrumental settings and electrical connections were readapted to functioning in MS-decoupled ESI regime of NanoMate system. The position of the electrospray chip was adjusted with respect to the HCT counterelectrode to ensure an optimal transfer of the ionic species into the mass spectrometer. The NanoMate can infuse the samples at every 10 seconds (high throughput regime). The BiVersa NanoMate system operating in Chip-Based Infusion mode utilizes the ESI Chip™ - a silicon-based integrated nanoelectrospray microchip device developed using deep reactive ion etching technologies. The ESI Chip consists of an array of 400 nanoelectrospray emitters with different inner diameters allowing for flow rates of 20 to 300 nL/min. The manufacturing process for the ESI Chip allows for consistent nozzles providing a robust and reliable platform. The nozzles on the ESI Chip provide a long, stable spray, and in infusion mode, eliminate sample-to-sample carryover.
The electrospray is initiated by applying a tension of 0.8 kV on the pipette tip and a pressure over the infusion tube of 0.6 p.s.i. after the sample infusion the tip is automatically ejected and it is replaced by a new one. The substances are loaded on a sample plate containing 96 orifices, the maximum volume of one of the orifices is 200 μL.
| Services: | TOP |
The mass spectrometry laboratory provides a complete structural analysis for different biological compounds with the identification of new substances and/or biomarkers present in small quantities in the sample.
| Contact: | TOP |
Head of Laboratory: