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INTRODUCTION

Alginates are polysaccharides from brown algae.

They form gels in the presence of divalent cations. These

gelling properties make them of interest for food industries

(thickening products, adhesive properties for restructured

meat or vegetables, foam stabilization), and in health

applications (wound healing, tissue engineering scaffolds

and implants).

The gelling property of alginates depends on the relative

proportion of guluronate homopolymeric blocks (poly-G).

A wide natural variability exists in the proportion of poly-G

blocks among seaweeds, as well as effects of

environmental and seasonal factors. It is therefore crucial

to develop analytical methods able to characterize finely

the structure of alginates, including the differentiation of

Guluronate(G) and Mannuronate (M) blocks, especially as

they cannot be differentiated by their mass and cannot be

easily separated by conventional LC systems coupled to

MS.

STRUCTURAL CHARACTERIZATION OF ALGINATE AND OTHER COMPLEX GLYCANS USING HIGH RESOLUTION ION MOBILITY - MASS SPECTROMETRY

Nick Tomczyk1; Laetitia Denbigh1; Helene Rogniaux2; David Ropartz2; Jakub Ujma1; Kevin Giles1 1 Waters Corporation, Wilmslow, UK, SK9 4AX, 2 INRA, UR1268 Biopolymers Interactions Assemblies, Nantes, France

EXPERIMENTAL

Samples Pure samples of β-D-Mannuronic acid oligomer, α-L-Guluronic acid oligomer (pure degree of polymerisation 5, see Figure 1) and an unknown sample which alternate β-D-Mannuronic acid and α-L-Guluronic acid randomly were diluted to 10µg/mL in 1:1 methanol:water. These diluted samples were analysed (on their own or as a mixture) in negative Ion ESI by infusion on a research MS platform comprising a modified SYNAPT HDMS system fitted with a prototype cyclic IMS device and dual gain ADC.

The cyclic IMS enabled research platform. This platform is based on a Synapt G2-Si (ESI-Q-IM-ToF) instrument and is shown schematically in Figure 2.

CONCLUSION

High resolution ion mobility with mass spectrometry was

shown as being an extremely powerful technique for the

detailed characterisation of these challenging compounds.

Rapid and easy to conduct experiments gave unique

valuable information, that no other analytical techniques

could obtain.

Homopolymeric blocks of consecutive G-residues (G-

blocks) or consecutive M-residues (M-blocks) were

separated. Contaminant with different conformations were

observed in the purified samples.

Multiple isomers were observed in the unknown mix

samples. Signature fragments unique to either Guluronate

or Mannuronate blocks helped with the finer

characterisation of the isomers.

OVERVIEW

Here we present methods to finely characterise complex polysaccharides using high resolution ion mobility (IM) in association with mass spectrometry. These methods will provide information on:

The contamination in purified samples The composition and arrangement of β-D-

mannuronate (M) and α-L-guluronate (G) blocks in mixed alginate samples.

These signature fragment ions were extracted in the mobil-

ity ATDs of the unknown sample C as shown in Figure 7.

The signature fragment characteristic of G blocks is mainly

present in isomers C3 and C4 whereas the signature frag-

ment characteristic of M blocks is mainly present in struc-

tures C2, C3 and C5. Other fragments were shown to be

characteristic of C1 and C6 or of C2, but their identity

needs to be confirmed as they are probably originating

from consecutive fragmentation. The finer characterisation

of the isomers could be improved by for example using O18

labelled samples to identify fragments generated by con-

secutive fragmentation.

Figure 2: Schematic of cyclic IMS enabled research platform.

The cyclic IM arrangement is shown in Figure 3. The cyclic IMS device and array provide a 100cm, single pass,

mobility path length offering IMS resolution of 60-70

By controlling the array region ions can be made to do further passes increasing the IMS path length by 1 meter per pass. In this way IMS resolution in excess of 200 are possible.

The cyclic IMS cell consists of a stacked ring ion guide arranged to give a circular ion path at 90° to the mass spectrometer ion path. Ions are controlled by manipulating the array region to “trap” “separate” or “eject ions for a specified time. Upon ejection, ions are returned back into the MS ion path. The number of passes around the cyclic mobility device is controlled by the set time delay between ion trapping and ejection. Either side of the array are 45mm long stacked ring ion guides (SRIGs) with axial fields applied to both transport ions or act as stores. At the entry to the high pressure region there is a standard He cell. Either side of the high pressure region are 15cm long SRIG devices with axial fields operating around 10-2 mb. The cyclic IM was operated at 1.8mb of N2 and T-Waves in the range 20to 40V at 375m/s-1. For more information on this Research platform please see poster TP385. Data Acquisition A 1:1mixture of pure β-D-Mannuronic acid and α-L-

Guluronic acid (DP5) was analysed using a 1, 3 and 6

passes of the cyclic IMS device (1,3 and 6 m of IMS). All

subsequent analyses shown here were done under same

conditions as for the 6 pass separation. The only exception

is when post IMS fragmentation was induced for fragment

ion generation.

Figure 3: Schematic of IMS region of research platform.

RESULTS

Separation of Parent ions. Figure 4 shows extracted ion (879.3 m/z) arrival time plots (EI-ATDs) for the separation of pure β-D-Mannuronic acid and α-L-Guluronic acid (DP5) for 1,3 and 6 passes of the cyclic IMS device.

Assuming a resolution of 65 for a single pass the

resolution for 6 passes would be approximately 160 . When infusions of the pure standards and mixed isomer samples are compared under the 6 pass conditions it is clear that β-D-Mannuronic acid has a significantly lower drift-time and therefore more compact structure compared to α-L-Guluronic acid as shown in Figure 5.

Figure 4: Extracted ion (879.3m/z) Mobility ATDs of 1:1 mix

of pure (A) β-D-Mannuronic acid and (B) α-L-Guluronic acid

(DP5) following 1(bottom), 3 (middle) and 6 (top) passes.

Pure samples and the mixed unknown isomer sample

were infused separately and analysed using 6 passes of the

cyclic IMS device. The ATDs are shown in Figure 5. Both

pure samples contained at least two isomers indicating

that sample purification was not perfect and that some

contamination occurred. Contamination could be semi-

quantified on the EI-ATDs traces. Comparison with

elementary composition obtained by other techniques

could validate the method for impurity semi-

quantification.

The unknown sample (C) mixes G and M blocks randomly. At least 6 different species are observed on the mobility ATDs. The main species C4 has the same arrival time as B2 indicating that pure α-L-Guluronic acid is the major component in sample C. Pure β-D-Mannuronic acid, does not seem to be present at a distinguishable level in the sample.

Figure 5: Extracted ion (879.3m/z) Mobility ATDs of pure (A) β

-D-Mannuronic acid, pure (B) α-L-Guluronic acid (DP5), and

unknown mixture (C).

Post IMS Fragmentation In addition to separation of parent ions, the 3 separa-tions shown in Figure 5 were followed by post IMS frag-mentation , which yields product ions that show same apparent drift-time as their precursor. The drift time aligned MS/MS spectra are shown in Figure 6 for the main peaks in pure samples A and B (peaks labelled A2 and B2 in Figure 5). Some fragment ions appear to be unique to β-D-Mannuronic acid (M blocks) or to α-L-Guluronic acid (G blocks). Some of these ions can be identified and be used as signature fragment ions for M or for G blocks. The signature fragment ions for M are mainly Z/C ions whereas signature fragment ions for G are mainly 0,2X-H2O ions.

Figure 6: Drift time aligned fragmentation spectra of peak A2

from pure (A) β-D-Mannuronic acid sample and peak B2

from pure (B) α-L-Guluronic acid sample.

Figure 7: Extracted ion Mobility ATDs in the mix sample (C) of

different signature fragment ions

Figure 1: Chemical structures of β-D-Mannuronic acid oli-

gomer (A) and α-L-Guluronic acid oligomer (B) (with

pure degrees of polymerisation 5)