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Characterization of heparin-like disaccharides using metallic cationization
Daniel Ortiz, Jeanine Tortajada, Jean-Yves Salpin
To cite this version:
Daniel Ortiz, Jeanine Tortajada, Jean-Yves Salpin. Characterization of heparin-like disaccharides us-ing metallic cationization. 27èmes journées françaises de spectrométrie de masse, Sep 2010, Clermont-Ferrand, France. �hal-01891872�
Experiments are carried out on a QSTAR Pulsar I (QqTOF) mass
spectrometer (Applied Biosystems/ MDS SCIEX) coupled to a nanospray ionization source.
All heparin-like disaccharides as well as copper and calcium salts were
purchased from Sigma.
1:1 binary mixtures of metallic salt and disaccharide (10-4 mol/L) were
prepared in pure water or in water/methanol (50/50).
MS/MS experiments with N2 as collision gas (P(N2)= 10-5 Torr).
CHARACTERIZATION OF ISOMERIC HEPARIN-LIKE
DISACCHARIDES USING METALLIC CATIONIZATION
D. Ortiz, J. Tortajada, J-Y. Salpin
Laboratoire Analyse et Modélisation pour la biologie et l’Environnement -Université d'Evry Val d'Essonne
Heparins and heparan sulfate are glycosaminoglycans (GAGS). They are composed of repeating disaccharide units made of hexuronic acids (1→4) linked to
glucosamine units (Figure 1). GAGs interact with a variety of proteins, and these interactions can have many physiological effects, notably on hemostasis, lipid transport or cell migration. Although tandem mass spectrometry has been successfully applied to oligasaccharide analysis, heparin analysis is still challenging for different reasons, notably because of the high density of sulfate groups, the pattern of which is an important determinant of the heparin bioactivity. Furthermore, sulfate groups are easily lost during CID experiments, resulting in the destruction of the structural information. In previous works, our group has shown that divalent metal ions can be successfully applied to oligosaccharide analysis and in this context we used this approach to differentiate heparin-like disaccharide isomers.
Introduction
Conclusion
1. JI. Capila and R.J. Linhardt, Angew. Chem. Int. Ed. 41, 391 (2002).
2. J-Y. Salpin, L. Boutreau, V. Haldys, J. Tortajada, Eur. J. Mass Spectrom. 7, 321 (2001 3. J.Y. Salpin, J. Tortajada, J. Mass Spectrom. 37, 379(2002).
4. L. Boutreau, E. Leon, J-Y. Salpin, B. Amekraz, C. Moulin, J. Tortajada, Eur. J. Mass
Spectrom. 9, 377 (2003)
5. A. El Firdoussi, M. Lafitte, J. Tortajada, O. Kone, J-Y. Salpin, J. Mass Spectrom. 42, 999 (2007)
Results and discussion
References
Perspectives
Fragments IIH + Cu IIIH + Cu
CHO X C2O X 49 X SO3 X CHO/SO3 X 0,2 X1 X 0,2 A2/SO3+H X X 0,2 X1SO3 X X 0,2 X1SO3/H2O X Y1/H2O X 273 X
Fragments IIA + Ca IIA + Cu
SO3 X X 0,2 X1 X X 0,2 X1/SO3 X X 0,2 X1/SO3/H2O X Z1/H2O X Z1/SO3 X C3H6O3 X 273 X
Figure 1: Different Mono sulfated-heparine isomers
Figure 2: ESI mass spectrum of a CaCl2/II-H mixture Figure 3: CID spectrum of the [Ca(II-H)]+complex
Figure 5: CID spectrum of the [Ca(III-H)]+complex
Table 2: CID comparison of [Cu(Hep)]+
Table 1: CID comparison of [Ca(Hep)]+
Table 3: CID comparison of [M(II-A)]+ Complexes [M(III-A)]+ not
observed, [M(III-A)-SO3]+
are formed.
CID mass spectra were recorded between 8 and 25 eV.
All fragment ions > 3% are included in tables 1-3.
Experiments with polysulfated heparin oligosaccharide.
Theoretical calculations on metal/heparin-disaccharide complexes in order to describe the effect of the complexation process onto the sugar unit. Alternative approaches (ternary complexes).
Experimental
We demonstrate that the combined use of metal cationization and tandem mass spectrometry can lead to successful distinction of mono sulfated heparin-like
disaccharides. Both calcium or copper dications indeed allow the position of the sulfate group to be located.
In the case of calcium salts (II-H/III-H isomers), we can observe that the calcium complexes generated by nanospray do not lose upon collision
the sulfate group while the copper dication induces desulfation. This finding is important in the context of the study of bigger multisulfated oligosaccharides.
O O O OH HO HOOC HO NHAc OH OSO3 O O O OSO3 HO HOOC HO NHAc OH OH II-A III-A O O O OH HO HOOC HO NH2 OH OSO3 O O O OSO3 HO HOOC HO NH2 OH OH II-H III-H
Figure 4: ESI mass spectrum of a CaCl2/III-H mixture
Fragments IIH + Ca IIIH + Ca
H20 X 0,2 A2 X X 0,2 A2/H2O X 0,2 X1 X 0,2 A2/C2H4O2 X X 0,2 A2/SO3 X 0,2 A2/C2H4O2/SO3 X X 0,2 A2/SO3/2H20 X 0,2 A2/C2H4O2/SO3/H2O X 0,4 A2 X m/z, amu 300 320 340 360 380 400 420 440 460 480 0 50 100 150 200 250 300 350 400 450 500 550 In te nsi ty , co un ts 456,00 396,91 438,00 426,51 395,99 336,94 378,95 0,2A 2 0,2A 2/H2O [Ca(III-H)]+ 0,4A 2 -H 2O III-H 300 320 340 360 380 400 420 440 460 480 m/z, amu 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2584 In te nsi ty , co un ts 396,91 455,94 426,44 417,44 339,94 379,00 437,93 316,93 0,2-A 2 [Ca(II-H)]+ 0,2X 1 II-H 0,2A 2/H2O
