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Heparin-like disaccharides: effect of metallic complexation in fragmentation pathways as characterized by MS/MS and UVPD experiments
Daniel Ortiz, Jean-Yves Salpin, Al Mokhtar Lamsabhi, Quentin Enjalbert, Manuel Yáñez, Philippe Dugourd
To cite this version:
Daniel Ortiz, Jean-Yves Salpin, Al Mokhtar Lamsabhi, Quentin Enjalbert, Manuel Yáñez, et al.. Heparin-like disaccharides: effect of metallic complexation in fragmentation pathways as characterized by MS/MS and UVPD experiments. SMAP 2011 (spectrométrie de masse et analyse protéomique), Sep 2011, Avignon, France. pp.213, 2011. �hal-00760099�
1
LAMBE UMR 8587, Laboratoire Analyse et Modélisation pour la Biologie et l’Environnement, Université d’Evry val d’Essonne, 91025 Evry (France).
2
Departament of Chemistry C‐9, Universidad autónoma de Madrid (UAM) Cantoblanco, 28049‐ Madrid (Spain).
3
Laboratoire de Spectrométrie Ionique et Moléculaire – LASIM – UMR 5579 ‐ Université Claude Bernard Lyon 1.
Heparin‐like disaccharides: effects of metallic complexation in fragmentation
pathways as characterized by MS/MS and UVPD experiments
D. Ortiz
1
, J-Y. Salpin
1
, A.M. Lamsabhi
2
, M. Yáñez
2
, Quentin Enjalbert
3
, Philippe Dugourd
3
Motivations :
Why Heparin disaccharides?Heparin (HP) glycosaminoglycan (GAGs)1, an anticoagulant drug, is recognized to be a
biologically important polysaccharide. It has been implicated in many biological processes such as blood coagulation, cell‐cell and cell‐matrix interaction inflammatory processes, cell growth, lipid transport and metabolism.
Why is it important to study the interaction between HP and metal cations?
The effect of metal ions on protein‐carbohydrate complexes is largely unknown. Heparin‐ biomolecule interaction can be influenced by the binding of metal ions to these complexes2.
For example, it has been reported that physiological Ca2+ induces conformational changes in
heparin that are necessary for the interaction between the anticoagulant Heparin and Annexin V, a protein proposed to play an important role in the inhibition of blood coagulation3. Then so, it is a Calcium‐depended interaction.
What is our strategy?
Experimentally, our aim was to study (Ca(II‐H))+ and (Ca(II‐A))+ complexes by tandem ESI/MS. Once generated in the gas phase, ions then undergo a fragmentation process by Collision Induced Dissociation (CID). Without metal there is no difference in the MS/MS spectra. Only
0,2A
2 fragmentation is observed. Ca2+ cation induces different conformational changes in both
isomers, resulting in completely different fragmentation pathway. Interaction between Acetyl/Ca2+ must be important in the dissociation process. Theoretically our aim is to explain this
Metal‐HP interaction by DFT calculations and delineate mechanisms of dissociation accounting for the experimental data.
Why UVPD photodissociation?
Ultraviolet photodissociation (UVPD) of positive and negative precursor ions appears to be
important activation method for providing complementary structural information to CID. UVPD favored additional cross‐ring cleavages of A and X type ion series, therefore enabling easier
sulfate group location.
.
MS/MS results:
Experiments were carried out on a LTQ Orbitrap
XL mass spectrometer coupled to an ESI source.
Nitrogen gas was used as collision gas
.
(Ca(II-H))+ 140 200 260 320 380 440 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R ela tive A bundance 397.08 456.08 426.58 376.17 438.08 340.08 (Ca(II-H))+ CID 12 0,2 A 2 * -SO3 - H2O 0,2 X1 ((Ca(II-H))2- 0.2A 2)+ : 150 200 250 300 350 400 450 500 550 600 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R ela tive A bundance 382.08 399.75 480.00 418.08 364.08 498.08 (Ca((II-A))+ -H2O 0,2 X 1 * +H2O -H2O (Ca(II-A))+ CID 15 -SO3 O O COOH HO OH O OH OH NH2 OSO3 0,2 A 2* O O COOH HO OH O OH OH NHAc OSO3 0,2 X 1* m/z
Computational results:
The geometries were optimized using the density functional theory (DFT) with the B3LYP hybrid functional and 6‐311G** basis set. Refined relative energies were obtained at 6‐
311++G(3df,2p) level. Without Ca2+ all the calculated 50 conformers, for each disaccharide, are
very close in energy (<50 KJ/mol). Nevertheless, when (Ca(HP))+ is formed it is observed an
increase in relative energies between conformers (>50KJ/mol).
High Binding Energy (BE) values (~1400 KJ/mol) are obtained. As deduced from the conformers calculation and the BE values, the metal complex stabilizes strongly one structure. It seems reasonable to deduce that both sugars lose partially their possibilities to change structurally.
Biologically, this consideration could be critical in order to explain the strong interactions
aforementioned. Analytically, when (Ca(HP))+ is formed, the molecule loses it flexibility due to
the fixation structure effect and therefore it is noticed a decrease in the number of fragments.
MS/UVPD results:
Experimental setup (below) consists of a LTQ‐quadrupole linear ion trap coupled to a visible/UV tunable OPO laser. A quartz window was fitted on the rear of the LTQ chamber to allow the introduction of the laser beam. Once the ions are isolated in the trap, they are
irradiated with the OPO laser (Panther OPO laser pumped by a 355‐nm Nd:YAG
Surelite;Continuum) and the photofragmentation mass spectra can be recorded.
Optical spectra (Left figure) were obtained recording UVPD spectra as a function of the laser
wavelength. Right figure shows the theoretical UV spectrum for (I‐H)2‐. It was obtained at
B3LYP/6‐311++G(2df,2p) level with TD(nstates=20).
CID and UVPD (at 240 nm) spectra for I‐H2‐ were also been recorded. Most of the fragment
ions resulting from CID were also observed in UVPD mode. As it was already reported4, additional cross‐ring fragments in UVPD mode (0,2A
2) provides more information about the
localization of sulfate group.
II‐H
II‐A
I‐H
(Ca(I‐H)‐H)
‐(I‐H)
2‐ Excitation Energy (nm)(Cu(I‐H)‐H)
‐ O scilla to r str eng th Excitation Energy (nm)(I‐H)
2‐ MS2 (I-H)2- CID 15 I‐H C1 Y1 H2O B1 200 205 210 215 220 225 230 235 240 245 250 255 260 m/z 0 10 20 30 40 50 60 70 80 90 100 R ela tive A bundance 217.98 247.50 222.01 238.49 254.98 258.03 202.50 236.97 0,2 A2 80 100 140 180 220 260 300 340 380 420 m/z 0 10 20 30 40 50 60 70 80 90 100 R ela tive A bundance 258.00 247.33 97.33 300.00 218.00 199.00 189.67 157.00 398.00 I‐H MS2 I-H ls240 0,2 A2 Y1 B1 HSO4‐ ‐HSO4 0.2X1 –SO3 0.2X1 2-B1 –SO3 2,4 A2 C1 O O COOH HO OSO3 O OH OH NH2 OSO3 O O COOH HO OSO3 O OH OH NH2 OSO3 Y1 0,2 A2 150 200 250 300 350 400 450 500 550 600 m/z 0 10 20 30 40 50 60 70 80 90 100 R ela tive A bundance 533.67 474.67 417.67 (Ca(I‐H)‐H)‐ 0,2 A 2 * 0,2X 1 * MS2 (Ca(I-H)-H)- ls220According to the II‐H/II‐A Calcium complexes, a decrease in the number of fragments is observed due to the metal stabilization (~200 KJ/mol). Nevertheless no difference have been reported between UVPD and CID fragmentation model
CID UVPD
UVPD
References:
1.I. Capila and R.J. Linhardt, Angew. Chem. Int. Ed., 391 (2002).
2.Y. Seo, M.R. Schenauer, J. A. Leary, Int. J. Mass Spectrom, 303, 191‐198 (2011).
3.I.Capila, M.J. Hernaiz, Y.D. Mo, T.R. Mealy, B. Campos, J.R. Dedman, R.J. Lindhardt, B.A. Seaton, Structure, 9, 57‐64, (2001).
4. A. Racaud, R. Antoine, L. Joly, N Mesplet, P. Dugourd, J. Lemoine, J. Am. Soc. Mass Spectrom, 20 1645‐1651 (2009).
