1. ESI ion trap mass spectrometry of
kappa-carrageenan
Hanne M. Devle & Dag Ekeberg
Agricultural University of Norway, IKBM, N-1432 Ås, Norway
INTRODUCTION
Carrageenan is a collective term for polysaccharides of about 25,000 galactose derivates extracted from red seaweed (Rhodophycae, Fig.1). These linear polymers have a regular but imprecise structures, dependent on the source and extraction conditions. Carrageenan is classified into three types as kappa (), iota () or lambda () carrageenan according to the number (one, two or three) of sulphate groups per repeat unit of disaccharide, respectively. -carrageenan consists of a repeating unit composed of the disaccharide, -(1-3)-D-galactose-4-sulfate and -(1-4)-3,6-anhydro-D-galactose (I).1
RESULTS AND DISCUSSION
In the mass spectrum of the tetrasaccharide of -carrageenan (Fig.3a), the base peak is found at m/z 394, which corresponds to the doubly charged anion (II). At m/z 811, the corresponding sodium adduct is observed. In the mass area of m/z 200–300, some fragment ions are visible. These ions have not been investigated but are believed to be present due to collision-induced dissociation (CID) in the ion trap of the product ions. These secondary fragments should not be of significant amounts in an ion trap. If the sample had contained significant amounts of desulphated tetrasaccharide, one would expect a signal at m/z 691. Desulphation of the analyte can take place at different times at different places during the analysis without the sample itself being desulphated. The CID spectrum of m/z 394 (Fig.3b) makes it clear that a significant amount of m/z 691 is present, which corresponds to the loss of HSO4-. The question is which of the two sulphate groups are eliminated. Scheme II illustrates the fragmentation pattern of MS2 of m/z 394. As expected, no secondary fragments are observed from this experiment. The next step in the procedure was to isolate m/z 691 and study the CID spectrum of this desulphated product ion, and obtain information of this structure. Scheme III illustrates two different sets of product ions depending on which sulphate group that is missing. In the MS3 spectrum of m/z 691 (Fig.3c), the product ion at m/z 241 identifies the actual structure. The remaining schemes and mass spectra, does not give any further solid structural information. I
Fig.1: Red seaweed (Rhodophycae)
AIM
The purpose of this work was to explore the possibility of using electrospray ionisation ion trap mass spectrometry (ESI-MS) to acquire detailed structural information for identification and characterisation of oligosaccharides of the type neocarrabiose. The starting point was the tetrasaccharide and the desire to investigate whether the collision energy in the ion trap would be sufficient in giving enough fragmentation to identify the position of the sulphate group in the molecule.
547
100 a
394 b c
529
394
529
Rel. Int. [%] 50
322
547
242
811
385
255
403
385
691
241
259
313
673
691
241
631
m/z
100
300
500
700
900
200
300
400
500
600
700
300
400
500
600
700
547
100
385
385 d e f
Rel. Int. [%] 50
529
241
403
241
193
241
260
385
223
449
139
153
181
305
EXPERIMENTAL
The tetrasaccharide of -carrageenan was obtained from the work by Ekeberg et.al, (2001)2, which included enzymic degradation and size exclusion chromatography. The ESI-MS system used was a Bruker Esquire ion trap (Fig.2). The analyte (0.01 mg/mL in 50/50 (v/v %) methanol and water) was introduced into the mass spectrometer by a Cole Parmer direct infusion pump, holding a constant flow of 0.2 mL/h. Both negative and positive ESI-MS modes have been attempted for the analysis of -carrageenan. As suspected, the negative ESI gave the best results and MS in the positive mode will therefore not be further discussed. The conditions during the ESI-MS were as follows: N2 was used as both drying and nebulizer gas. The nebulizer pressure was kept at 15 psi, the dry gas flow at 5 L/min and the drying temperature at 300 C. The capillary voltage held 3270 V and the capillary exit was at -121 V.
m/z
200
300
400
500
200
300
400
500
150
200
250
300
350
Fig.3: MSn spectra of the tetrasaccharide of -carrageenan. a) n=1, b) n=2 m/z 394, c) n=3 m/z 691, d) n=3 m/z 547, e) n=3 m/z 529 and f) n=4 m/z 385 II
IIIa
IIIb
IVa
IVb Va Vb
VIa
VIa
Fig.2: The Bruker Esquire ion trap
CONCLUSION
ESI-MS and MSn are techniques that show promise with regards to obtaining structural information from small amounts of the oligosaccharide of -carrageenan. The ion trap does not seem to be appropriate further than to MS4, due to loss of sensitivity. Some product ions have been observed during MS4, which can be explained as secondary fragments. However, this should not take place.
REFERENCES
Yoshiaki, Y., Thuy, T.T.T., Urakawa, H. and Kajiwara, K. Food Hydrocolloids 16 (2002)
Ekeberg, D., Knutsen, S.H. and Sletmoen, M. Carbohydrate Research 334 (2001)