Method for sequencing oligosaccharides

Abstract

The present invention concerns a method for sequencing oligosaccharides, which makes it possible to identify the primary sequence of an oligosaccharide of unknown structure, including its monosaccharide composition, the position (regiochemistry) and configuration (stereochemistry) of glycosidic bonds, the nature and position of functional modifications, and its branched structure, particularly including the identification of the reducing end.

Claims

1. A method for sequencing oligosaccharides, wherein it comprises the steps of i. fragmentation of the oligosaccharides into disaccharides and monosaccharides while preserving the molecular structure of the constituents as present in the oligosaccharide to be sequenced ii. separation of each previously obtained disaccharide and monosaccharide by mass spectrometry, iii. analysis by infrared (IR) vibrational spectroscopy of each previously separated disaccharide and monosaccharide, iv. identification of the structure of each disaccharide and monosaccharide by comparison of the obtained IR spectra with a set of reference disaccharide and monosaccharide IR spectra, and v. definition of the oligosaccharide sequence by combination of the structures identified for each disaccharide and monosaccharide.

2. The method according to claim 1, wherein the fragmentation of the oligosaccharides into disaccharides and monosaccharides (step i.) is done by mass spectrometry.

3. The method according to claim 1, wherein the fragmentation by mass spectrometry is done by CID, CAD, SID, ETD, ECD and laser-induced fragmentation.

4. The method according to claim 1, wherein the IR spectroscopy is performed at a wavelength ranging from 4000 to 2000 cm.sup.1.

5. The method according to claim 1, wherein the IR spectroscopy is done by the IRMPD method implemented in an ion trap.

6. An apparatus for sequencing oligosaccharides comprising a mass spectrometry device, an electromagnetic radiation source, a database and processing means, wherein it comprises a processor for controlling the steps of i. fragmentation of the oligosaccharides into disaccharides and monosaccharides while preserving the molecular structure of the constituents as present in the oligosaccharide to be sequenced ii. separation of each previously obtained disaccharide and monosaccharide by mass spectrometry with the mass spectrometry device, iii. analysis by infrared (IR) vibrational spectroscopy of each previously separated disaccharide and monosaccharide with the electromagnetic radiation source, iv. identification of the structure of each disaccharide and monosaccharide by comparison of the obtained IR spectra with a set of reference disaccharide and monosaccharide IR spectra contained in the database, and v. definition of the oligosaccharide sequence by combination of the structures identified for each disaccharide and monosaccharide by the processing means.

7. The apparatus according to claim 6, wherein the electromagnetic radiation source is a L.A.S.E.R. source.

8. The apparatus according to claim 6, wherein the electromagnetic radiation source is integrated into the mass spectrometry device.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a schematic representation of the implementation of the method according to the invention: (i) fragmentation of the oligosaccharide by mass spectrometry, (ii) separation of each of the fragments preparatory to IR spectroscopy analysis, (iii) IR spectroscopy analysis, (iv) identification of the structure of each constituent monosaccharide and disaccharide by comparison with the reference IR spectra contained in the database DB, (v) definition of the sequence by the combinatorial method described in the invention.

(2) FIGS. 2, 3 and 4 refer to the implementation of the method on the example of the tetrasaccharide GlcN(1.fwdarw.4)GlcNAc(1.fwdarw.4)GlcNAc(1.fwdarw.4)GlcNAc

(3) FIG. 2 shows the production of the set of constituent monosaccharide fragments (labelled a, b, c, d) and disaccharide fragments (labelled a-b, b-c, c-d) of the tetrasaccharide (labelled 0) by several mass spectrometry steps MS.sup.n.

(4) FIG. 3 shows the analysis of each of the monosaccharide fragments by IRMPD spectroscopy (top boxes) and their comparison with the reference spectra with which the fragment spectra were identified (bottom boxes).

(5) FIG. 4 shows the analysis of each of the disaccharide fragments by IRMPD spectroscopy (top boxes) and their comparison with the reference spectra with which the fragment spectra were identified (bottom boxes).

EXAMPLES

(6) 1. Materials and Methods

(7) Materials

(8) The materials used to implement the method of the invention include a commercial ion-trap mass spectrometer equipped with an electrospray ion source (Thermofinnigan LCQ). This device is modified to allow the injection of an infrared L.A.S.E.R. beam generated by a YAGpumped tunable OPO/OPA system (LaserVision) at a rate of 10 Hz. It is notably described in Schindler, B. et al. (Phys. Chem. Chem. Phys. 2014, 16, 22131-22138)

(9) Fragmentation

(10) Fragmentation of the samples is done by the CID method, in several successive fragmentation steps if need be.

(11) Sampling and IR Spectroscopy

(12) The method of sample preparation by MS.sup.n and IRMPD spectroscopy is that described in Schindler, B. et al. (Phys. Chem. Chem. Phys. 2014, 16, 22131-22138).

(13) 2. Analysis of a Tetrasaccharide

(14) FIGS. 2, 3 and 4 refer to the implementation of the previously described method on the example of the tetrasaccharide
GlcN(1.fwdarw.4)GlcNAc(1.fwdarw.4)GlcNAc(1.fwdarw.4)GlcNAc

(15) All structural information obtained by comparison of the fragment spectra and the reference spectra is listed in Table 1.

(16) TABLE-US-00001 TABLE 1 Identification of the structure of each disaccharide and monosaccharide by comparison of the obtained IR spectra with a set of reference disaccharide and monosaccharide IR spectra. Fragment MS.sup.n m/z Generic type identification identification of monosaccharide structure a MS.sup.4 180 HexN GlcN b MS.sup.4 222 HexNAc GlcNAc c MS.sup.3 204 HexNAcH.sub.2O GlcNAcH.sub.2O d MS.sup.2 222 HexNAc GlcNAc identification of disaccharide structure a-b MS.sup.3 383 HexNHexNAc GlcN (1.fwdarw.4) GlcNAc b-c MS.sup.3 407 HexNAcHexNAcH.sub.2O GlcNAc (1.fwdarw.4) GlcNAcH.sub.2O c-d MS.sup.2 425 HexNAcHexNAc GlcNAc (1.fwdarw.4) GlcNAc

(17) The oligosaccharide sequence is then obtained by combination of the structural information:

(18) ##STR00001##

(19) The structure obtained is indeed that of the tetrasaccharide GlcN(1.fwdarw.4)GlcNAc(1.fwdarw.4)GlcNAc(1.fwdarw.4)GlcNAc.

REFERENCES

(20) Both, P. et al., Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing. Nat. Chem. 2013, 6, 65-74. Gaye, M. M. et al., Multidimensional Analysis of 16 Glucose Isomers by Ion Mobility Spectrometry. Anal. Chem. 2016, 88, 2335-2344. Nagy, G.; Pohl, N. L. B., Monosaccharide identification as a first step toward de novo carbohydrate sequencing: Mass spectrometry strategy for the identification and differentiation of diastereomeric and enantiomeric pentose isomers. Anal. Chem. 2015, 87, 677-685. Nagy, G.; Pohl, N. L. B., Complete Hexose Isomer Identification with Mass Spectrometry. J. Amer. Soc. Mass Spectrom. 2015, 26, 677-685. Schindler, B. et al., Distinguishing isobaric phosphated and sulfated carbohydrates by coupling of mass spectrometry with gas phase vibrational spectroscopy. Phys. Chem. Chem. Phys. 2014, 16, 22131-22138 Stefan, S. et al., Differentiation of Closely Related Isomers: Application of Data Mining Techniques in Conjunction with Variable Wavelength Infrared Multiple Photon Dissociation Mass Spectrometry for Identification of Glucose-Containing Disaccharide Ions. Anal. Chem. 2011, 83, 8468-8476