1) The document describes the development of a new FRET probe using the fluorescent proteins Lumazine and Venus linked by a protease-specific cleavage sequence. 2) It characterizes the FRET efficiency between Lumazine and Venus and develops substrates sensitive to thrombin and MMP-2 enzymes. 3) Results show the probe's emission spectrum and anisotropy change upon cleavage, and it can detect protease activity in solution and linked to a hydrogel using confocal microscopy, demonstrating its potential for enzymatic studies.
MASS SPECTROMETRY IN THE FIELD OF FOOD INDUSTRYErin Davis
This is a powerpoint presentation solely to give a brief idea about the role of Mass Spectrometry (MS) which is one of the powerful analytical technique.This presentation describes the role of Mass Spectrometry in the field of food industry.These slides deals with the basic principle,working,components,detailed analysis etc.
Dr.S.Karthikumar
Asst. Prof., Dept. of Biotechnology
Kamaraj College of Engineering and Technology
S.P.G.C.Nagar, Virudhunagar, Tamilnadu, India
skarthikumar@gmail.com
48 Measurement of the Σ beam asymmetry for the ω photo-production off the pro...Cristian Randieri PhD
Measurement of the Σ beam asymmetry for the ω photo-production off the proton and the neutron at GRAAL - June 2013
di V. Vegna, A. D'Angelo, O. Bartalini, V. Bellini, J. P. Boquet, M. Capogni, L. E. Casano, M. Castoldi, F. Curciarello, V. De Leo, J. P. Didelez, R. Di Salvo, A. Fantini, D. Franco, G. Gervino, F. Ghio, G. Giardina, B. Girolami, A. Giusa, A. Lapik, P. Levi Sandri, A. Lleres, F. Mammoliti, G. Mandaglio, M. Manganaro, D. Moricciani, A. Mushkarenkov, V. Nedorezov, C. Randieri, D. Rebreyend, N. Rudnev, G. Russo, C. Schaerf, M. L. Sperduto, M. C. Sutera, A. Turinge, I. Zonta (2013)
Abstract
We report on new measurements of the beam asymmetry for ω photo-production on proton and neutron in Hydrogen and Deuterium targets from the GRAAL collaboration. The beam asymmetry values are extracted from the reaction threshold (E = 1.1 GeV in the free nucleon kinematics) up to 1.5 GeV of incoming photon energy. For the first time both the radiative and the three- pion decay channels are simultaneously investigated on the free proton. Results from the two decay channels are in agreement and provide important constraints for the determination of resonant state contributions to the ω production mechanism. First experimental results on the deuteron allow the extraction of the _ beam asymmetry on quasi-free nucleons. Comparison of the results for free and quasi-free kinematics on the proton shows a generally reasonable agreement, similar to the findings in pseudo-scalar meson photo-production reactions. For the first time measurements on quasi-free neutrons are available, showing that both the strength and the angular distributions of the beam asymmetry are sensibly different from the results on the proton target.
MASS SPECTROMETRY IN THE FIELD OF FOOD INDUSTRYErin Davis
This is a powerpoint presentation solely to give a brief idea about the role of Mass Spectrometry (MS) which is one of the powerful analytical technique.This presentation describes the role of Mass Spectrometry in the field of food industry.These slides deals with the basic principle,working,components,detailed analysis etc.
Dr.S.Karthikumar
Asst. Prof., Dept. of Biotechnology
Kamaraj College of Engineering and Technology
S.P.G.C.Nagar, Virudhunagar, Tamilnadu, India
skarthikumar@gmail.com
48 Measurement of the Σ beam asymmetry for the ω photo-production off the pro...Cristian Randieri PhD
Measurement of the Σ beam asymmetry for the ω photo-production off the proton and the neutron at GRAAL - June 2013
di V. Vegna, A. D'Angelo, O. Bartalini, V. Bellini, J. P. Boquet, M. Capogni, L. E. Casano, M. Castoldi, F. Curciarello, V. De Leo, J. P. Didelez, R. Di Salvo, A. Fantini, D. Franco, G. Gervino, F. Ghio, G. Giardina, B. Girolami, A. Giusa, A. Lapik, P. Levi Sandri, A. Lleres, F. Mammoliti, G. Mandaglio, M. Manganaro, D. Moricciani, A. Mushkarenkov, V. Nedorezov, C. Randieri, D. Rebreyend, N. Rudnev, G. Russo, C. Schaerf, M. L. Sperduto, M. C. Sutera, A. Turinge, I. Zonta (2013)
Abstract
We report on new measurements of the beam asymmetry for ω photo-production on proton and neutron in Hydrogen and Deuterium targets from the GRAAL collaboration. The beam asymmetry values are extracted from the reaction threshold (E = 1.1 GeV in the free nucleon kinematics) up to 1.5 GeV of incoming photon energy. For the first time both the radiative and the three- pion decay channels are simultaneously investigated on the free proton. Results from the two decay channels are in agreement and provide important constraints for the determination of resonant state contributions to the ω production mechanism. First experimental results on the deuteron allow the extraction of the _ beam asymmetry on quasi-free nucleons. Comparison of the results for free and quasi-free kinematics on the proton shows a generally reasonable agreement, similar to the findings in pseudo-scalar meson photo-production reactions. For the first time measurements on quasi-free neutrons are available, showing that both the strength and the angular distributions of the beam asymmetry are sensibly different from the results on the proton target.
Join Brian Searle on an illustrated tour about interpreting MS/MS peptide spectra. On this tour you will first see how you can relate mass spectra to peptides. Next you see why the SEQUEST software was developed to interpret these spectra as peptides. Next you will see other software approaches have been developed and how combining approaches produces even better results.
19 Double π0 Photoproduction on the Proton at GRAAL - Physical Review Letters...Cristian Randieri PhD
Double π0 Photoproduction on the Proton at GRAAL - The American Physical Society, Physical Review Letters, June 2003, Vol. 90, N. 22, pp. 222001-1-222001-4, ISSN: 0031-9007, doi: 10.1103/PhysRevLett.90.222001
di Y. Assafiri, O. Bartalini, V. Bellini, J. P. Bocquet, S. Bouchigny , M. Capogni, M. Castoldi, A. D'Angelo, J. P. Didelez, R. Di Salvo, A. Fantini, L. Fichen , G. Gervino, F. Ghio, B. Girolami, A. Giusa, M. Guidal, E. Hourany, V. Kouznetsov, R. Kunne, J. M. Laget, A. Lapik, P. Levi Sandri, A. Lleres, D. Moricciani, V. Nedorezov, D. Rebreyend, C. Randieri, F. Renard, N. Rudnev, C. Schaerf, M. Sperduto, M. C. Sutera, A. Turinge, A. Zucchiatti (2003)
Abstract
The double π0 photoproduction off the proton has been measured in the beam energy range of 0.65 1.5GeV. The total and differential cross sections and the Σ beam asymmetry were extracted. The total cross section measured for the first time in the third resonance region of the nucleon shows a prominent peak. The interpretation of these results by two independent theoretical models infers mostly the selective excitation of P11- and D13-nucleon resonances.
Carlos Afonso, Université de Rouen, Laboratoire COBRA, Plateau technique C2iorga
In this presentation, Carlos Afonso describes the analysis of polymers and petroleum by ion mobility mass spectrometry and utilises novel sample introduction techniques such as the Atmospheric Solids Analysis Probe (ASAP).
45 Evidence for a narrow N* (1685) resonance in quasifree Compton scattering ...Cristian Randieri PhD
Evidence for a narrow N* (1685) resonance in quasifree Compton scattering on the neutron - The American Physical Society, Physical Review C, February 2011, Vol. 83, N. 2, pp. 022201-1-022201-4, ISSN: 0556-2813, doi: 10.1103/PhysRevC.83.022201
di V. Kuznetsov, M. V. Polyakov, V. Bellini, T. Boiko, S. Chebotaryov, H.-S. Dho, G. Gervino, F. Ghio, A. Giusa, A. Kim, W. Kim, F. Mammoliti, E. Milman, A. Ni, I. A. Perevalova, C. Randieri, G. Russo, M. L. Sperduto, C. M. Sutera, A. N. Vall (2011)
Abstract
The study of quasifree Compton scattering on the neutron in the energy range of Eγ=0.75–1.5 GeV is presented. The data reveal a narrow peak at W~1.685 GeV. This result, being considered in conjunction with the recent evidence for a narrow structure at W~1.68 GeV in η photoproduction on the neutron, suggests the existence of a nucleon resonance with unusual properties: a mass M~1.685GeV, a narrow width Γ⩽30 MeV, and the much stronger photoexcitation on the neutron than on the proton.
In this paper, student projects are given as an example on how to introduce FT –NMR into the undergraduate curriculum.
We will incorporate NMR experiments that illustrate the application of high resolution NMR spectroscopy to the structure determination of Anti-Cancer agents.
High resolution 13 C and 1H NMR , 13 C –distortionless enhancement by polarization transfer (DEPT) , 2D 13C-1H correlated (HECTOR), and 2D 1H-1H correlated (COSY) spectroscopy techniques will be used for elucidating skeletal arrangement of monomer units
Fluorescence spectroscopy is a very advanced technology that uses the phenomena of fluorescence. This presentation covers the basic concepts, instrumentation, applications, advantages and disadvantages of the technique. It also covers the Jablonski diagram. The process that analyses and measure these types of emissions is known as Fluorescence spectroscopy.Fluorescence spectroscopy is a novel technique that is used for measuring the binding of ligands to the proteins in the presence of fluorphore that bound to the ligand .
Join Brian Searle on an illustrated tour about interpreting MS/MS peptide spectra. On this tour you will first see how you can relate mass spectra to peptides. Next you see why the SEQUEST software was developed to interpret these spectra as peptides. Next you will see other software approaches have been developed and how combining approaches produces even better results.
19 Double π0 Photoproduction on the Proton at GRAAL - Physical Review Letters...Cristian Randieri PhD
Double π0 Photoproduction on the Proton at GRAAL - The American Physical Society, Physical Review Letters, June 2003, Vol. 90, N. 22, pp. 222001-1-222001-4, ISSN: 0031-9007, doi: 10.1103/PhysRevLett.90.222001
di Y. Assafiri, O. Bartalini, V. Bellini, J. P. Bocquet, S. Bouchigny , M. Capogni, M. Castoldi, A. D'Angelo, J. P. Didelez, R. Di Salvo, A. Fantini, L. Fichen , G. Gervino, F. Ghio, B. Girolami, A. Giusa, M. Guidal, E. Hourany, V. Kouznetsov, R. Kunne, J. M. Laget, A. Lapik, P. Levi Sandri, A. Lleres, D. Moricciani, V. Nedorezov, D. Rebreyend, C. Randieri, F. Renard, N. Rudnev, C. Schaerf, M. Sperduto, M. C. Sutera, A. Turinge, A. Zucchiatti (2003)
Abstract
The double π0 photoproduction off the proton has been measured in the beam energy range of 0.65 1.5GeV. The total and differential cross sections and the Σ beam asymmetry were extracted. The total cross section measured for the first time in the third resonance region of the nucleon shows a prominent peak. The interpretation of these results by two independent theoretical models infers mostly the selective excitation of P11- and D13-nucleon resonances.
Carlos Afonso, Université de Rouen, Laboratoire COBRA, Plateau technique C2iorga
In this presentation, Carlos Afonso describes the analysis of polymers and petroleum by ion mobility mass spectrometry and utilises novel sample introduction techniques such as the Atmospheric Solids Analysis Probe (ASAP).
45 Evidence for a narrow N* (1685) resonance in quasifree Compton scattering ...Cristian Randieri PhD
Evidence for a narrow N* (1685) resonance in quasifree Compton scattering on the neutron - The American Physical Society, Physical Review C, February 2011, Vol. 83, N. 2, pp. 022201-1-022201-4, ISSN: 0556-2813, doi: 10.1103/PhysRevC.83.022201
di V. Kuznetsov, M. V. Polyakov, V. Bellini, T. Boiko, S. Chebotaryov, H.-S. Dho, G. Gervino, F. Ghio, A. Giusa, A. Kim, W. Kim, F. Mammoliti, E. Milman, A. Ni, I. A. Perevalova, C. Randieri, G. Russo, M. L. Sperduto, C. M. Sutera, A. N. Vall (2011)
Abstract
The study of quasifree Compton scattering on the neutron in the energy range of Eγ=0.75–1.5 GeV is presented. The data reveal a narrow peak at W~1.685 GeV. This result, being considered in conjunction with the recent evidence for a narrow structure at W~1.68 GeV in η photoproduction on the neutron, suggests the existence of a nucleon resonance with unusual properties: a mass M~1.685GeV, a narrow width Γ⩽30 MeV, and the much stronger photoexcitation on the neutron than on the proton.
In this paper, student projects are given as an example on how to introduce FT –NMR into the undergraduate curriculum.
We will incorporate NMR experiments that illustrate the application of high resolution NMR spectroscopy to the structure determination of Anti-Cancer agents.
High resolution 13 C and 1H NMR , 13 C –distortionless enhancement by polarization transfer (DEPT) , 2D 13C-1H correlated (HECTOR), and 2D 1H-1H correlated (COSY) spectroscopy techniques will be used for elucidating skeletal arrangement of monomer units
Fluorescence spectroscopy is a very advanced technology that uses the phenomena of fluorescence. This presentation covers the basic concepts, instrumentation, applications, advantages and disadvantages of the technique. It also covers the Jablonski diagram. The process that analyses and measure these types of emissions is known as Fluorescence spectroscopy.Fluorescence spectroscopy is a novel technique that is used for measuring the binding of ligands to the proteins in the presence of fluorphore that bound to the ligand .
We investigated the gas‐phase fragmentation reactions of a series of 2‐aroylbenzofuran derivatives
by electrospray ionization tandem mass spectrometry (ESI‐MS/MS). The most intense fragment
ions were the acylium ions m/z 105 and [M+H–C6H6]+, which originated directly from the
precursor ion as a result of 2 competitive hydrogen rearrangements. Eliminations of CO and CO2
from [M+H–C6H6]+ were also common fragmentation processes to all the analyzed compounds.
In addition, eliminations of the radicals •Br and •Cl were diagnostic for halogen atoms at aromatic
ring A, whereas eliminations of •CH3 and CH2O were useful to identify the methoxyl group
attached to this same ring. We used thermochemical data, obtained at the B3LYP/6‐31+G(d) level
of theory, to rationalize the fragmentation pathways and to elucidate the formation of E, which
involved simultaneous elimination of 2 CO molecules from B.
Lightoptical nanoscopy for the use in biomedical applications and material sciences, detection in attomolar concentrations
* Use of standard fluorophores like GFP, RFP, YFP, Alexa, Fluorescein (no photoswitch necessary)
2CLM Two Color Localisation microscopy in the nanoscale
* Optical resolution 10 nm in 2D, 40 nm in 3D
* Very fast in processing, complete picture (2000 images) with processing in 3 minutes
Fluorescence spectroscopy becomes a widely used tool at the interface of biology, chemistry and physics, because of its precise sensitivity and recent technical advancements. The measurements can provide information on a wide range of molecular processes including the interactions of solvent molecules with fluorophores, rotational diffraction of biomolecules, distance between sites of biomolecules, conformational changes and binding interactions. These advances in fluorescence technology are decreasing the cost and complexity of previously complex processes. Fluorescence spectroscopy is a highly developed and non-invasive technique that enables the on-line measurements of substrate and product concentrations or the identification of characteristic process states.
JNLIV Supplement Quantum Response of Human Skin to Hydrogen Peroxide Stimulation
chrisNellPosterA0
1. Development of new genetically encoded FRET probe
based on the fluorescent proteins Lumazine and Venus
MASTER IN BIOENGINEERING, Christophe Nell
In the laboratory of Professor Gerard Marriott, University of California Berkeley,
Supervised by Dr. Alexander Hoepker, UC Berkeley.
Under the direction of Prof. Philippe Renaud, EPFL
Materials and methods
Introduction
Results
Discussion
Reference Acknowledgements
The aim of this project is to study a new fluorescent recombinant protein composed of Lumazine protein (LumP) and Venus. The overlap spectrum between the
emission of LumP under excitation at 400nm and the absorption of Venus produce an efficient Förster Resonance Energy Transfer (FRET) phenomenon. I used this
system to develop new substrates for thrombin and MMP-2. Thrombin is a well know enzyme that allows us to characterize the efficiency of the transfer of energy
related to the cleavage[1]
. We know that the efficiency of the transfer (E) of energy is related to the distance R0
[2]
:
R0 can be experimentaly calculated with the overlap integral (J) between the emission of the donor LumP and the acceptor Venus.
n is the refractive indext of the middle, k² the orientation factor of the probe, ΦD the absolute quantum yield of the donor.
With the data recorded with LumP and Venus and the software flurotools[3]
, we obtain Ro = 52 angstroms. We recorded the change of emission spectrum and the
change of anisotropy during the cleavage of the substrates. The recombinant protein sensitive to MMP-2 was created because the level of this enzyme is a key
diagnosis for metastasis[4,5]
. Finally, the substrats were also used with a hydrogel to detect the activity of the enzyme with living cells using a confocal microscope.
3 differents substrats were produced to study this new recombinant protein: the first
without cleavage sequence and only 2 amino acids between the two proteins, the
second with 11 amino acids including -LVPR/GS- the sequence sensitive to thrombin,
and finally 13 amino acids including the MMP-2 substrate -IPVS/LRSG.
To record the change of emission spectrum, we used a PTI (Photon Technology
International) fluorometer and the software FelixGX 4.1.2. We recorded the change of
emission spectrum by looking at the emission intensity of LumP and Venus under
excitation at 400nm.
For the anisotropy, we used the luminescent spectrometer AB2 (AMINCO Bowman
Serie 2). The sample was excited and the emission intensity corresponding to the peak
of Lump was recorded with filters for the excitation and emission light. Then, the
anisotropy R can be calculated by the formula:
Finally, to record the cleavage of the probe with a hydrogel, we used a confocal
microscope.
3 different cell lines were cultivated on the new hydrogel: Hep-G2, NBT-2 and MDA-
MB-231. We did an overnight incubation before taking the images used to calculate the
amount of FRET on the surface of the gel.
On the left, the plasmid for the MMP-2
substrate. On the right, picture of the
bacteria BL21 expressing the
recombinant protein LumP-Venus taken
with the confocal microscope under
excitation at 405nm with a filter that
keeps only the light after 510nm.
Before the cleavage, the emission is maximal
at 530nm. After the cleavage, the emission is
maximal at 470nm, which is the peak of
emission of LumP.
V stands for Verical, the filter at 0° and H for a filter at 90°. The factor G
is a corrector factor for the instrument.
We have a 4.51-fold change for the ratio between the peak of emission of LumP and
Venus before and after the injection of thrombin. Similar results were recorded with the
MMP-2 sensitive substrate during the emission spectrum.
To track the cleavage of the recombinante proteins with the hydrogel, we illuminated it
with a 405nm laser. Then, we recorded the emission in two channels, before and after
510nm in order to separate the emission of LumP and Venus. To find out the activity of
MMP-2, we looked at the difference of ratio between the emission of LumP and Venus.
The graph on the left shows the different intensity and the ratio related to the yellow
line on the picture taken by the confocal microscope.
Emission spectrum recorded with
10μM of substrate and 14.7nM of
thrombin. The first recording at t0
is done without enzyme and the
curve in orange is done 18min
after the injection of the enzyme.
On the right, the ratios
470/530nm calculated from the
same experiment.
The anisotropy is calculated with the same
concentration than the emission spectrum. We
can see a decrease of the anisotropy from 0.245
to 0.166. It decreases because the cleavage
reduces the time between the absorption of the
photon and the emission of light. Moreover, a
smaller molecule tumble faster. This is why LumP
alone has a lower anisotropy.
On the left picture, MDA-MB-231 cells, objective
20x. The results of the Hep-G2 cells and the
NBT-2 cells are similar with no correlation
between a decrease of intensity in C2 related to
an increase of intensity in C1.
Because no real difference have been observed with the
cells, we produced a hydrogel with thrombin substrate
linked on the top of it. Then the enzyme was injected
directly into the buffer to mimick the action of MMP-2.
We can see that even if we loose emission intensity in
both channels, we have a significant change of ratio.
[1] Zhang, B., 2004. Design of FRET-based GFP probes for detection of protease inhibitors. Biochemical and Biophysical Research Communications,
323(2), pp.674–678.
[2] Valeur, B., 2001. Molecular Fluorescence: Principles and Applications Wiley-VCH. Available at: http://www.citeulike.org/group/2000/article/2395562
[3] a|e - UV-Vis-IR Spectral Software 1.2, FluorTools,, Available at: www.fluortools.com
[4] Yang, J. et al., 2007. Detection of MMP activity in living cells by a genetically encoded surface-displayed FRET sensor. Biochimica et biophysica acta,
1773(3), pp.400–7. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17187878 [Accessed October 27, 2014].
[5] B. Packard, V. Artym et al., 2009. Direct visualization of protease activity on cells migrating in three-dimensions. Matrix Biol., 28(1), pp.3–10. Available
in PMC 2010 January 1.
[6] Piston, D.W. & Kremers, G.-J., 2007. Fluorescent protein FRET: the good, the bad and the ugly. Trends in biochemical sciences, 32(9), pp.407–14.
Available at: http://www.ncbi.nlm.nih.gov/pubmed/17764955 [Accessed July 11, 2014].
The recominant protein are linked to a 4-arm PEG SH/
Mal using MBS. Fibronectin with Traut's reagent is also
used to help the cells to stick on the gel.
We proved that the pair LumP-Venus separated by a protease specific sequence can be used for enzymatic study in the cuvette. However, the results with
the hydrogel did not allow to detect the protease activity of the cells. The lost of fluorescence on the gel during the cleavage by trombin in the two channels
shows that both, the stability of the protein and the attachement on the gel need to be improved. With the cells, no significant cleavage is detected as we
expect a change of ratio with an decrease of emission in the second channel C2 that is similar than for the emission scan with MMP-2 done in a cuvette.
Nevertheless, we can say that this new recombinant protein is suitable for enzymatic detection. The cleavage can be followed with different strategies and
this sustrates have different advantages compare to the probe currently used[6,4]
.
- The link between LumP and Venus can easily be changed and the recombinant proteins can be produced at low cost.
- The change of ratio between the peak of emission of LumP and Venus reaches 4.5-fold with 11 amino acids which is better than the usual pair CFP-YFP.
- The cleavage can easily be detected over time with an emission scan and the anisotropy of LumP.
- The change ratio for the emission intensity of LumP and Venus can be detected with a confocal microscope.
Emission scan before (t0)
and after the injection of
MMP-2
Prof. Gerard Marriott, who accepted me in his laboratory at UC Berkeley.
Dr. Alexander Hoepker, my supervisor during this project., who has taught me all the
techniques necessary to achieve this project. Moreover, the entire laboratory deserves a
special thank for the welcome and all the help that they personally provided me.
Prof. Heinis, who generously offered a stock solution of MMP-2 produced in his laboratory
of therapeutic proteins and peptides (LPPT) at EPFL.
Prof. Philippe Renaud, my supervisor from EPFL.
My family and my friends, old and new, who gave me support and made my stay a great
experience.
Cleavage of the substrats on
the hydrogel by thrombin