Septembre 2011 Issue 90 I www.analis.be

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Septembre 2011 Issue 90 I NEW Microtiterplate Automation Microwave Sample Prep Cell Analysis & Flow Cytometry WORKSHOP October 2011 Ultracentrifugation NEW Rotor NEW CESI-MS Extending the Reach of MS NEW Chemiluminescence & Fluorescence Imaging Systems
  • quantification de l'adn par uv
  • cesi-ms
  • mass spectrometry
  • high capacity
  • protein signals
  • sheath-liquid
  • capillary electrophoresis
  • using
Publié le : mardi 27 mars 2012
Lecture(s) : 73
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Source : analis.be
Nombre de pages : 48
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NEW Chemiluminescence
Septembre 2011 Issue 90 I www.analis.be & Fluorescence Imaging Systems
NEW CESI-MS
Extending the Reach of MS
Ultracentrifugation
NEW Rotor
Cell Analysis & Flow Cytometry
WORKSHOP October 2011
Microwave Sample Prep
NEW Microtiterplate AutomationQuantifcation de l’ADN par UV/Vis
Maintenant aussi spécifque que le Picogreen™ *
*Picogreen est une marque commerciale d’Invitrogen by Life Technologies
1 Pipetter l’échantillon
dans la plaque DropPlate
2 Mesurer l’absorbance
avec le DropSense 96 3 Quantifcations spécifques
(96 échantillons en 5 minutes) de l’ADN et des contaminants
90
A
Septembre 2011
Vous pouvez télécharger
nos Flashmag à l'URL:
www.analis.be/pubs.
asp?cid=213&lcid=29
Coordination
Eliane Henri : ehi@analis.be
B
Production
Comparison between DNA quanti- Sylvie Mathelot
fcation methods. (A) Comparative Séverine Firla
analysis between most common
used DNA quantifcation methods
Tirageusing samples from different
origins. (B) Sample content analysis
2 890 exemplairesusing cDrop software with dsDNA,
RNA and protein as references.
Diffusion
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Microwave Sample Prep
27 Single Reaction Chamber (SRC) - Digestion in a benchtop package I MILESToNE
Septembre 2011
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Synthesis Equipment
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37 Enceinte d’essais climatiques pour essais de stabilité selon GMP et ICH I MEMMERT
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Product viewEliane Henri : ehi@analis.be
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Have a look at some of our scientifc publications at www.beckmancoulter.com/wsrportal/eventDetails/GLB_BCI_147852
Analis
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notre laboratoire de recherche AnALIS r&D Diag
développe, depuis plus de 25 ans, des kits et méthodes
d’analyse utilisant les techniques d’électrophorèse et
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ceofx). Analis r&D Diag dispose d’une unité de recherche Analis R&D Diag Laboratory sera présent au:
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Capillary ElectrophoresisCes kits sont destinés aux laboratoires de biologie
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MEDICA 2011AnALIS r&D DIAg est toujours à la recherche
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www.analis.be>products>CE-capillary electrophoresis
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Innovations in
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With special topics on
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in the Flow Cytometry Process
Analis, in collaboration with Beckman Coulter,
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Please let us know if you are interested in receiving
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www.beckmancoulter.com/CESI
Un système d’électrophorèse capillaire spécialement conçu pour le
couplage à la spectrométrie de masse (MS)
Le champ d'application de la spectrométrie de masse technologies telles que la LC-MS à phase inversée, sont
va pouvoir être étendu grâce à la combinaison de maintenant bien visibles grâce au CESI 8000.
l’électrophorèse capillaire à haute performance et du
“Le CESI 8000 a été conçu en collaboration avec des
nouveau module ESI de Beckman Coulter. Les débits ultra
chercheurs en spectrométrie de masse qui utilisent
faibles atteints par ce système augmentent la sensibilité
plusieurs applications différentes, et qui cherchaient à
de la spectrométrie de masse et tout en réduisant le
étendre la gamme d’analytes détectés et à augmenter
phénomène de suppression d’ions.
la sensibilité globale” explique Jeff Chapman, director of
Orange County, Calif. – (le 2 juin 2011) Beckman Coulter Discovery Products. “Le résultat est un
Le CESI 8000 High-Performance Separation-ESI Module système de séparation « frontend » qui génère des données
avec la Technologie OptiMS de Beckman Coulter, Inc. complémentaires à HPLC et qui fournit aux chercheurs
comprend le premier atomiseur CESI commercialement d’importantes informations qui n’étaient pas disponibles
disponible en combinaison avec le nouveau système auparavant.
d’électrophorèse capillaire conçu spécialement pour
Le passage aisé de LC-ESI-MS à la CESI-MS permet
la spectrométrie de masse (MS). Ainsi, les chercheurs
l’intégration de CESI dans les processus d’analyse existants.
disposent d’une interface puissante et très sensible entre
Le CESI 8000 est monté sur une paillasse mobile, réglable
l’électrophorèse capillaire (CE) et la MS, et en même
en hauteur pour pouvoir l’ajuster rapidement à la hauteur
temps d’une gamme d’analytes étendue complémentaire
du spectromètre de masse. Des adaptateurs ‘plug-and-
à LC-MS et GS-MS.
spray’ existent déjà pour toutes les grandes marques de
La technologie optiMS se caractérise par une séparation spectrométrie de masse.
CE à débit extrêmement bas (jusqu’à 10 nl/min) qui est
combiné avec l’ionisation par électronébulisation (ESI) au
cours d’un seul et unique processus dynamique dans
le capillaire même. On peut donc parler de CESI. Pour
accomplir la CESI, l’extrémité du capillaire doit être poreuse
au fux ionique de façon à fermer le circuit électrophorétique
tout en permettant d’entamer le processus ESI dans le
même appareil.
La CESI-MS génère des séparations de peptides avec
des pics élevés (> 320) ce qui permet d’analyser des
échantillons très complexes au niveau protéomique. Les
débits ultrafaibles utilisés en CE, réduisent effectivement la
suppression d’ions et permet de détecter des modifcations
post-traductionnelles (PTMs) comme la phosphorylation et
la sialylation qui typiquement gênent l’ionisation. Grâce à
l’effcacité et la sensibilité des séparations, il est possible
de détecter des peptides qui ne sont présents qu’en
très faibles quantités, à savoir dans des concentrations
picomoléculaires. En plus, la sélectivité de la CE dans
des séparations « frontend », permet la caractérisation
de protéines intactes, de leurs isoformes, de fragments
clivés et de complexes.
Le CESI 8000 avec la Technologie OptiMS de Beckman
Coulter permet également une séparation rapide et
effcace de composants pharmaceutiques, métabolites
pharmaceutiques, acides organiques, acides aminés,
amines à faible poids moléculaire, peptides, acides
nucléiques et nucléosides. Des molécules souvent
pas remarquées ou pas du tout détectables avec les www.analis.be>products>capillary electrophoresis
Info : Luc Van Laer I Tél. 09 243 77 10 I lvl@analis.be Capillary Electrophoresis
CESI-MS of Intact Proteins
Introduction or liquid junctions. In the CeSI design (Figure 1), the last 3-4
cm of the bare fused-silica capillary are etched with hydrofuoric
there is a growing need for selective and sensitive analytical acid until the section becomes conductive, producing an ~5-μm
tools for the characterization of intact proteins. the effective thick porous wall, which is conductive when in contact with an
coupling of capillary electrophoresis and electrospray ionization electrolyte. Capillaries with porous tips are simply inserted in a
time-of-light mass spectrometry (Ce-eSI-toF-MS) may offer an stainless steel eSI needle flled with a static conductive liquid.
attractive option by providing both the separation and detection
In this study, the performance of the CeSI-MS of intact proteins required to distinguish structurally-related protein species [1,2].
was evaluated using some model proteins. reproducibility, In order to bring Ce-eSI-MS to a high performance level, novel
linearity and LoDs were established. It is demonstrated that sheathless Ce-MS interfacing was studied to achieve sensitive
CeSI-MS provides enhanced responses for intact proteins detection of intact proteins.
resulting in sub-nM detection limits. this is a 50- to 140-fold
based on a design of Moini [3], a prototype CeSI sprayer was improvement compared to conventional sheath-liquid Ce-MS
recently developed in the laboratories of beckman Coulter. interfacing using the same capillary dimensions.
Flow rates in Ce are very low and the initial droplets formed during
the sheathless electrospray process are small, which leads to
Materials and Methodsmore effcient ionization (i.e., nanospray). With sheathless [1]
interfacing, the eSI [2] spray tip can be positioned close to the
Chemicals
MS inlet, thereby improving ion sampling effciencies. overall,
Acetic acid (99.8%), ammonium hydroxide (25%) and iso-this can lead to enhanced sensitivity and lower limits-of-
propanol (IPA) were obtained from Merck. Insulin (bovine detection (LoDs). the CeSI sprayer provides electrical contact
pancreas), carbonic anhydrase II (bovine erythrocytes), without the need for metal conductive coating, microelectrodes
ribonuclease A (bovine pancreas) and lysozyme (chicken egg
white) were from Sigma-Aldrich. Protein test mixtures were
prepared in the appropriate concentration with deionized water.
A bge of 100 mM acetic acid (pH 3.1) containing 5% IPA was
prepared by diluting 0.171 mL glacial acetic acid to 30 mL
with deionized water/IPA (95/5, v/v) and adjusting the pH with
ammonium hydroxide.
CE system
experiments were carried out on a beckman Coulter capillary
electrophoresis instrument. the separation voltage was -30 kV
and the capillary temperature was 20°C. Fused-silica capillaries
(total length, 100 cm; inner diameter, 30 μm, outer diameter,
150 μm) equipped with the porous tip [3] (length, 3-4 cm) were
supplied by beckman Coulter. the capillaries were precoated
with polyethylenimine (PeI). At the beginning of each day, the
coated capillary was conditioned by fushing the capillary at 50
psi with air (10 min), methanol (20 min), deionized water (5 min)
and bge (10 min). before each run, the capillary was fushed for
3 min (50 psi) with fresh bge. the sample was injected for 10
s at 5 psi.
CE-MS
MS detection was performed using a bruker Daltonics microtoF
orthogonal-accelerated time-of-f ight (toF) mass spectrometer.
Sheathless CE-MS
the capillary with the porous tip was placed in a grounded
stainless steel needle that could be positioned by an XyZstage
(beckman Coulter) ftting the bruker microtoF instrument (Figure
1B). A nanospray end plate and gas diverter were installed to
allow nanoeSI. the porous tip protruded the grounded needle
approximately 0.5 cm and the needle was flled with bge to Figure 1. (A) Detailed representation of the CESI sprayer design.
establish the electrical contact. the optimized spray conditions (B) Schematic representation of the CESI-MS set-up using the porous tip.
www.analis.be>products>capillary electrophoresis
Info : Luc Van Laer I Tél. 09 243 77 10 I lvl@analis.be Biotech 90 7Capillary Electrophoresis
were as follows; dry gas temperature, 180°C;
dry gas nitrogen fow, 3.0 L/min; nebulizer
pressure, 0.0 bar. electrospray in positive
ionization mode was achieved using an eSI
voltage of -2.1 kV.
Sheath-liquid CE-MS
For sheath-liquid interfacing, the capillary with
porous tip was placed in a grounded coaxial
Ce-MS sprayer (Agilent t echnologies) installed
on a conventional eSI source comprising the
standard eSI endplate and capillary cap. A fow
of 2 μL/min of IPA-water-acetic acid (75:25:0.1,
v/v/v) was applied as sheath liquid. the
optimized conditions for sheathliquid interfacing
were: dry gas temperature, 180°C; dry gas
nitrogen fow, 4 L/min; nebulizer pressure, 0.4
bar; eSI voltage, -4.0 kV. Figure 2. (A) BPE obtained with sheathless CESI-MS of a mixture of insulin (1), carbonic
anhydrase II (2), ribonuclease A (3) and lysozyme (4) (each 5 μg/mL). (B) Mass spectra
Data analysis obtained at the apices of peaks 1-4. Further conditions, see Materials and Methods Section.
CeSI-MS data were analyzed using bruker
concentration and obtained peak areas were obtained. Table 1 Daltonics Data Analysis software. base-peak electropherograms
also lists the LoDs (S/n=3) achieved with sheathless CeSI-MS. (bPe) were constructed in the range m/z 1000-3000. For
Sub-nM levels could still be detected for carbonic anhydrase II, determination of detection linearity and LoD, extracted-ion
ribonuclease A and lysozyme indicating very favorable LoDs for electropherograms (eIe) for the four model proteins were
CeSI-MS of intact proteins.constructed from their most abundant m/z signals. these were
m/z 1147.7 and 1434.1 for insulin, m/z 1210.4, 1262.9, 1320.3,
c dsheathless interface sheath liquid interface1383.1 and 1452.2 for carbonic anhydrase II, m/z 1521.2,
1711.1 and 1955.5 for ribonuclease A, and m/z 1431.6, 1590.34 2 2protein R LOD R LOD
and 1789.0 for lysozyme. insulin 0.999 1.28 0.992 106
carbonic 0.989 0.58 0.981 79
Results and Discussion anhydrase II
ribonuclease A 0.992 0.62 0.989 33
the porous tip capillary was placed in a grounded needle that
lysozyme 0.997 0.50 0.990 41was positioned on an XyZ-stage (Figure 1B). the electrical
contact was established by flling the stainless steel needle with
Table 1. Linearity (R2)a and LODs (nM)b for the four model proteins
bge. the liquid hardly evaporated from the needle and the same
obtained with CESI-MS and CE-MS with sheathliquid interfacing.
liquid could be used for an entire day of measurements. the
a. Concentration range, 0.05-25 μg/mL (CESI-MS) and 1-100 μg/mL (sheath liquid);
porous tip capillaries were coated with positively-charged PeI,
b. to yield S/N ratio of 3 as calculated by extrapolation from 1-μg/mL
which exhibits a net positive charge when an acidic bge is used. injection. For carbonic anhydrase analyzed with sheath-liquid CE-MS, a 5-μg/mL
injection was used ; c. BGE, 100 mM ammonium acetate (pH 3.1) containing 5% under these conditions, adsorption of peptides and proteins to
(v/v) isopropanol ; d. BGE, 100 mM ammonium acetate (pH 3.1).the capillary wall will generally be avoided due to electrostatic
repulsion. Most stable electrospray formation and highest analyte In order to assess the gain in signal provided by CeSI-MS, a
signals were obtained with the capillary tip at a distance of 3 mm comparison with sheath-liquid Ce-MS was made. the same
to the MS inlet. capillary, i.e. equipped with the porous tip, was placed in a
coaxial sprayer installed on a conventional eSI source. A sheath A mixture of insulin, carbonic anhydrase II, ribonuclease A and
liquid of IPA-water-acetic acid (75/25/0.1, v/v/v) was applied lysozyme was analyzed by sheathless CeSI-MS. using a bge of
at a fow rate of 2 μL/min. these conditions had shown to be 100 mM acetic acid (pH 3.1) with 5% IPA, the four proteins were
optimal for intact protein analysis in a previous Ce-MS study [4]. baseline separated within 11 min (Figure 2A). IPA was added to
A baseline separation within 10 min (Figure 3) and linear protein the bge as it provided a modest gain in protein signal intensities.
signals (Table 1) were obtained for the test proteins, indicating good quality mass spectra were obtained under these conditions
the proper functioning of the sheath-liquid interfacing.(Figure 2B). Deconvolution of the mass spectra yielded masses
of 5733.6 Da (insulin), 29024.6 Da (carbonic anhydrase II),
Figure 3. BPE obtained
13681.8 Da (ribonuclease A) and 14304.5 Da (lysozyme), with sheath-liquid CE-MS
respectively, which agreed well with their expected molecular of a mixture of insulin (1),
masses. the overall performance of the sheathless CeSI-MS carbonic anhydrase II (2),
system was further evaluated by assessing the repeatability, ribonuclease A (3) and
detection linearity and LoDs for the test proteins. Migration time lysozyme (4) (each 50 μg/
mL) using a conventional rSDs for all four proteins were less than 0.8% (n=5) and peak
ESI source and a sheath area rSDs were within 8% (n=5). Plate numbers ranged from
liquid of isopropanol-
0.5 x 105 (insulin) up to 1.5 x 105 (ribonuclease A). Clearly, the
water-acetic acid
CeSI-MS system allows repeatable and effcient analyses of (75/25/0.1, v/v/v). Further
intact proteins. t o check for detection linearity, protein mixtures conditions, see Materials
with concentrations between 0.05 and 25 μg/mL of each protein and Methods Section.
were prepared and each solution was analyzed in triplicate. For
all proteins, good linear relationships (Table 1) between injected
www.analis.be>products>capillary electrophoresis
8 Biotech 90 Info : Luc Van Laer I Tél. 09 243 77 10 I lvl@analis.be Capillary Electrophoresis
Protein signals obtained with sheath liquid interfacing were
approximately a factor 10 to 50 lower with respect to CeSI-MS (cf.
fgures 2A and 3 ; note that injected protein concentrations are 5
and 50 μg/mL, respectively). In particular, insulin showed reduced
response with sheath liquid Ce-MS. the lower protein signals are
the overall result of the dilution of the Ce effuent by the sheath liquid
and the lower ionization and ion sampling effciencies obtained using
conventional eSI in stead of nanospray. It should be noted that the
IPA in the sheath liquid strongly enhances protein ionization, as
without IPA, protein signals are even much lower. Furthermore, the
baseline noise observed is more favorable in CeSI-MS (Figure 4).
the application of sheath liquid causes signifcant chemical noise,
which is avoided with CeSI-MS. overall, the LoDs achieved with
CeSI-MS are considerably lower than for sheath-liquid Ce-MS Figure 4. Baseline signal and noise in the 0.9-5.9 min interval
(Table 1). during (A) CESI-MS and (B) sheath-liquid CE-MS of the protein test
mixture. Traces represent extracted-ion traces for insulin (1; m/z
1434.1), carbonic anhydrase II (2; m/z 1262.9), ribonuclease A (3;
Conclusion m/z 1711.1) and lysozyme (4; m/z/ 1590.3). Further conditions, see
Materials and Methods Section.
the performance of CeSI-MS for the analysis of intact proteins was
evaluated. Sub-nanomolar LoDs were obtained with the CeSI-
1 2 1MS system using a nanoeSI source. these are highly favorable Rob Haselberg , Chitra K. Ratnayake , Gerhardus J. de Jong , Govert
sensitivities which, to our knowledge, have not been achieved before 1W. Somsen
with Ce-MS for intact proteins. the gain in performance of the CeSI-
1. biomolecular Analysis, utrecht university, universiteitsweg 99, 3584 Cg,
MS system with respect to sheath-liquid Ce-MS can be attributed
the netherlands I 2. beckman Coulter, Inc., 250 S. kraemer blvd, M/S
to the reduced noise levels and increased analyte responses. the C1.nW.05, brea, CA 92822, united States
very favorable LoDs and overall performance indicates that CeSI-
ReferencesMS can be highly useful for intact protein analysis.
[1] r. Haselberg, g.j. de jong, g.W. Somsen, j. Chomatogr. A, 1159 (2007)
81; [2] r. g.j. de jong, g.W. electrophoresis, 32 (2011)
66 ; [3] M. Moini, Anal. Chem. 79 (2007) 4241 ; [4] r. Haselberg, g.j. de jong,
g.W. Somsen, Anal. Chim Acta, 678 (2010) 128.
CESI-MS, Peer Reviewed Publications
Bibliography 1st August 2011
1) characterization of drug-lysozyme conjugates by sheathless capillary
electrophoresis–time-of-fight mass spectrometry. R. Haselberg, S. Harmsen,
M.E.M. dolman, G.J. de Jong, R.J. kok and G.W. Somsen. Analytica chimica
Acta 698 (2011) 77– 83.
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electrophoresis-mass spectrometry. Rawi Ramautar, Jean-Marc Busnel, André
M. deelder, and oleg A. Mayboroda. Anal chem – In Review
3) optimization and Evaluation of a Sheathless capillary Electrophoresis-
Electrospray Ionization-Mass Spectrometry (cE-ESI-MS) Platform for Peptide
Analysis: comparison to Lc-ESI-MS. klaus Faserl, Bettina Sarg, Leopold kremser
and Herbert Lindner, Anal chem – In Press
4) Performance of a sheathless porous tip sprayer for capillary electrophoresis
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5) High capacity capillary electrophoresis-electrospray ionization mass spectrometry:
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6) Metal displacement and stoichiometry of protein-metal complexes under native
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mass spectrometry using a porous tip. Nguyen
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