JP5097608B2 - Identification method of sugar chain sequence - Google Patents

Description

The present invention predicts the m / z value of a fragment ion in an MS ² spectrum obtained when mass spectrometry is applied to chondroitin (CH) or chondroitin sulfate (CS) at an arbitrarily set sugar chain length and sulfate group position. Regarding the method. Furthermore, the present invention is obtained using the m / z value of the fragment ion in the MS ² spectrum obtained by subjecting CH or CS with unknown sugar chain length and sulfate group position to mass spectrometry, and the above prediction method. Further, the present invention relates to a method for identifying the unknown sugar chain length and sulfate group position by comparing the m / z values.

The abbreviations used in this specification and the like are described below.
CH: Chondroitin CS: chondroitin sulfate GAG: glycosaminoglycan GAG drug: sugar discovery was a starting point the GAG MS: Mass spectrometry MS ^n: multi-stage mass spectrometry MS ^2: 2-step mass spectrometry Hep: Heparin HS: heparan GalNAc sulfate: N-acetylgalactosamine GlcA: glucuronic acid CSA: chondroitin sulfate A (GalNAc in which the hydroxyl group at the C-4 position is sulfated (GalNAc4S))
CSC: Chondroitin sulfate C (sulfurized at hydroxyl group C-6 of GalNAc (GalNAc6S))
ESI-IT: Electrospray ionization-Ion trap type CID: Collision induced dissociation

    In the sugar chain structure analysis, a multi-stage mass spectrometer has been frequently used in recent years. Even in the GAG research scene, which has a particularly complex structure among sugar chains, MS has been used vigorously since the late 1990s (Non-patent Document 1).

    The difficulty in structural analysis of GAG is due to the fact that the molecular structure can exist indefinitely due to the presence of structural isomers (sulfate group positional isomers and uronic acid epimers). In order to realize GAG drug discovery, we will elucidate structures essential for controlling biological functions from sequences containing isomers that can exist indefinitely, and based on the molecular structure information, It is necessary to design a new arrangement that can artificially control the above. And, in the GAG drug discovery work, structural analysis technology contributes at a much higher weight compared to the case where a general low molecular compound or protein is used as a drug discovery candidate compound (non-patent document). 2).

The identification of sulfate group positional isomers, which are often attracting particular attention in the structural analysis of GAG by MS ⁿ , has so far been based on short-chain oligosaccharides (unsaturated disaccharides obtained by degrading GAG with a degrading enzyme). Many have been implemented on subjects. For example, non-patent literature 3 and non-patent literature 4 can be cited as examples of CS disaccharide analysis, and non-patent literature 5 can be cited as an example of analysis of Hep / HS disaccharide. Non-patent document 6 is given as an example of identifying uronic acid epimers.

On the other hand, although there is an attempt to make maximum use of MS ⁿ for the purpose of GAG structural analysis, it can be said that a highly universal method has not been developed yet. So far, an algorithm for analyzing the molecular structure of Hep / HS oligosaccharide by MS ⁿ has been published (Non-Patent Document 7), but structural information on the uronic acid epimer cannot be obtained using this method. Non-Patent Document 8 introduces a method for analyzing the sequence of keratan sulfate, but it is a methodology that cannot be applied to GAG having uronic acid.

    Among GAGs, in particular, CS has been in the limelight for many years because of its unique structure and physiological activity. CS also has many isomeric structures and high sequence heterogeneity (Non-patent Document 9, Non-patent Document 10 and Non-patent Document 11).

Several attempts to analyze the sequence of CS by MS ⁿ have also been reported, but it can be pointed out that they all lack decisive power.

For example, Non-Patent Document 12 and Non-Patent Document 13 identify structural isomers of CSA and CSC based on the ionic strength of the MS ² spectrum, and show that the same rule applies to long-chain oligosaccharides. In the description in the literature, the oligosaccharide sample itself used for obtaining standard data is considered to be a mixture of sequence isomers, and the error range of ionic strength is not clearly shown, which may cause difficulties in sequencing accuracy. . In Non-Patent Document 14, an MS ² spectrum of a highly sulfated long-chain oligosaccharide is obtained with high sensitivity, but the binding position of the sulfate group has not been identified.

  Furthermore, although Non-Patent Document 4 presents a sequence analysis method using a CS-decomposing enzyme together, it is limited to estimating the chain length of an alternating sequence, a random sequence or a block sequence.

Under these circumstances, a method for determining the CS sequence with higher accuracy by using MS ² with high analytical sensitivity and high throughput is awaited.
Trends in Glycoscience and Glycotechnology 18, 293-312, 2006 Medical Science Digest 33,964-965,2007 Journal of American Society of Mass Spectrometry 11,916-920,2000. Analytical Chemistry 73, 3513-3520, 2001 Journal of American Society of Mass Spectrometry 15, 1274-1286, 2004 Journal of American Society of Mass Spectrometry 18, 234-244, 2007 Analytical Chemistry 77, 5902-5911, 2004 Analytical Chemistry 78, 891-900, 2006 Advances in Pharmacology 53, 33-48, 2006 Advances in Pharmacology 53,282-295,2006 Advances in Pharmacology 53,523-539,2006 Analytical Chemistry 73, 6030-6039, 2001 Journal of American Society of Mass Spectrometry 14, 1270-1281, 2003 Electrophoresis 25, 2010-2016, 2004

An object of the present invention is to provide a method for predicting the m / z value of a fragment ion in an MS ² spectrum obtained when CH or CS in which a sugar chain length and a sulfate group position are set is subjected to mass spectrometry. is there. A further object of the present invention is to provide a method for easily identifying the sugar chain length and sulfate group position of CH or CS whose molecular structure is unknown by mass spectrometry.

As a result of analyzing each MS ² spectrum for high-purity CH oligosaccharide, CSA oligosaccharide and CSC oligosaccharide, the present inventors have found that regular fragment ions are generated for each structure. . And it discovered that the m / z value in the MS ² spectrum of the fragment ion could be predicted by a predetermined formula based on the sugar chain length and sulfate group position of CH, CSA and CSC oligosaccharides. Furthermore, the predicted value of the m / z value in the MS ² spectrum of CH and CS at an arbitrarily set sugar chain length and sulfate group position, and the m / z value of CH and CS whose sugar chain length and sulfate group position are unknown By comparing and comparing the measured values, it was found that the sugar chain length and sulfate group position of the unknown CH and CS can be identified, and the present invention has been completed.

That is, the present invention
(1) A method of predicting an m / z value in an MS ² spectrum of a fragment ion generated when chondroitin or chondroitin sulfate is subjected to MS ² including at least the following steps:
(I) a step of setting a sugar chain length and a sulfate group position of chondroitin or chondroitin sulfate consisting of a disaccharide unit that is a target of prediction of m / z value in MS ² spectrum;
(II) a step of selecting a disaccharide unit to which a glycosidic bond cleaved when the chondroitin or chondroitin sulfate of (I) is subjected to MS ² ;
(III) Steps for determining the following numerical values of (i) to (iv) for chondroitin or chondroitin sulfate of (I); and (i) N: number of disaccharide units,
(Ii) u: the number of the disaccharide unit selected in the above (II) when the disaccharide unit is numbered as one unit in order from the reducing end,
(Iii) s: the number of sulfate groups of the fragment ion generated by cleavage of the glycosidic bond belonging to the disaccharide unit selected in (II) above,
(Iv) a: the valence of the fragment ion generated by the cleavage of the glycosidic bond belonging to the disaccharide unit selected in (II) above,
(IV) Each numerical value obtained in the step (III) was selected in the above (II) by applying to the following formulas (A) to (D) according to any of the following (A) to (C): Determining an m / z value in the MS ² spectrum of a fragment ion selected from B, C, Y and Z-types that can be generated by cleavage of a glycosidic bond belonging to a disaccharide unit;
(A) When N-acetylgalactosamine constituting the disaccharide unit to which the glycosidic bond selected in (II) above does not have a sulfate group, the following formulas (b) and (d):
(B) When it has a sulfate group at the C-4 position of the N-acetylgalactosamine, the following formulas (a) and (c), or (C) the C-6 position of the N-acetylgalactosamine All of the following formulas (a) to (d)
(A) B _{2 (Nu) +1} ion (m / z) = {[379 (N−u) +176] +80 sa−a} / a,
(B) C _{2 (Nu) +1} ion (m / z) = {[379 (N−u) +176] + 18 + 80s−a} / a,
(C) Y _2u-1 ion (m / z) = {[379 (u-1) +203] + 18 + 80s-a} / a,
(D) Z _2u-1 ion (m / z) = {[379 (u-1) +203] + 80s-a} / a,
(2) A method for identifying the sugar chain length and sulfate group position of chondroitin or chondroitin sulfate with unknown molecular structure, comprising at least the following steps;
(V) A step of attaching chondroitin or chondroitin sulfate of unknown molecular structure to MS ² to obtain m / z values of all fragment ions observed in the obtained MS ² spectrum,
(VI) All fragments that can be produced by cleaving chondroitin or chondroitin sulfate with the sugar chain length and sulfate position set to MS ² by the method described in (1), by cleaving the glycosidic bond in the disaccharide unit Obtaining a predicted value of m / z value in the MS ² spectrum for ions;
(VII) All m / z values obtained by the step (V) were compared with the predicted values of all m / z values obtained by the step (VI). Sometimes, the step of identifying the sugar chain length and sulfate group position of chondroitin or chondroitin sulfate with unknown molecular structure as the sugar chain length and sulfate group position set in the step (VI),
(3) Until all the m / z values acquired in the step (V) and the predicted values of all m / z values acquired in the step (VI) substantially match (VI) And the identification method according to (2), further comprising the step of repeating the step of (VII),
(4) A method for identifying the sugar chain length and sulfate group position of chondroitin or chondroitin sulfate with unknown molecular structure, comprising at least the following steps;
(VIII) When each of a plurality of chondroitin or chondroitin sulfates composed of disaccharide units set at different sugar chain lengths and sulfate positions by the method described in (1) is subjected to MS ² , The predicted value of m / z value in the MS ² spectrum is obtained for all fragment ions that can be generated by the cleavage of the glycosidic bond, and the correspondence between the sugar chain length and sulfate group position and the predicted value of m / z value Creating a database about
(IX) subjecting chondroitin or chondroitin sulfate of unknown molecular structure to MS ² to obtain m / z values of all fragment ions observed in the obtained MS ² spectrum;
(X) All m / z values acquired in the step (IX) are compared with m / z values in the database created in the step (VIII), and all m / z values acquired in the step (IX) are obtained. The sugar chain length and sulfate group position of chondroitin or chondroitin sulfate having a predicted value of m / z value substantially matching the m / z value of
(5) The method according to any one of (1) to (4), wherein the chondroitin or chondroitin sulfate is chondroitin oligosaccharide or chondroitin sulfate oligosaccharide, respectively.
Is to provide.

The present invention has an excellent effect that the sugar chain length and sulfate group position of CH and CS can be easily identified only by using general mass spectrometry data. Specifically, a predicted value of the m / z value obtained when CH and CS in which the sugar chain length and the sulfate group position are set is attached to MS ² as a database, and the predicted value of this m / z value, It is possible to easily and quickly identify the sugar chain length and sulfate group position of CH and CS by collating with the actual measurement value of the m / z value in an actual sample containing CH or CS whose molecular structure is unknown. It is also possible to provide a structural analysis application by inputting regularity of ion fragmentation by MS ² of CH and CS into commercial software.

    Hereinafter, embodiments of the present invention will be described in detail.

    The sugar chains to be analyzed by the method of the present invention are CH and CS. CH and CS, together with hyaluronic acid and the like, are included in the category of sugar chains called GAG, and exist as the surface of animal cells including humans and cartilage components, and control various functions in the living body. It is attracting attention as a molecule.

    The structure of CH and CS has a linear basic skeleton in which two types of monosaccharides (GalNAc and GlcA) are alternately repeated by glycosidic bonds, and can be regarded as a repeating structure of disaccharide units composed of GalNAc and GlcA. Can do. Those having no sulfate group are CH, and those having a sulfate group are CS. The CS disaccharide unit has various isomers depending on the position of the sulfate group, but typical examples include CSA and CSC. The disaccharide unit is a single sulfate group position isomer (sulfuric acid). And those composed of different types of sulfate group positional isomers (including those not containing sulfate groups).

    In this specification and the like, CSA having a uniform arrangement refers to CS in which the disaccharide units constituting the CSA are composed of only CSA.

    In the present specification and the like, a CSA oligosaccharide having a uniform sequence refers to an oligosaccharide in which the disaccharide unit constituting the CSA oligosaccharide is composed only of CSA, and is also simply referred to as a CSA oligosaccharide.

    In this specification and the like, CSC having a uniform arrangement refers to CS in which the disaccharide units constituting the CSC are composed of only CSC.

    In the present specification and the like, the CSC oligosaccharide having a uniform sequence refers to an oligosaccharide having a disaccharide unit comprising only CSC, and is also simply referred to as a CSC oligosaccharide.

    In this specification and the like, the CH oligosaccharide refers to an oligosaccharide in which the disaccharide unit constituting it consists only of CH.

CH or CS capable of predicting the m / z value in the MS ² spectrum by the method of the present invention has an even number of monosaccharides constituting them, and a GalNAc residue at the reducing end. It is what you have. The sugar chain length is not particularly limited, and usually the number of disaccharide units constituting the CH or CS is an integer of 1 to 10, and the number of disaccharide units is 1 to 9, 1 to 4, 1 to An integer of 3 can also be exemplified. In the present invention, such CH or CS is referred to as CH or CS composed of disaccharide units.

    CH or CS capable of identifying the sugar chain length and sulfate group position according to the present invention has an even number of monosaccharides constituting them, and has a GalNAc residue at the reducing end. Is. The sugar chain length is not particularly limited, and usually the number of disaccharide units constituting the CH or CS is an integer of 1 to 10, and the number of disaccharide units is 1 to 9, 1 to 4, 1 to An integer of 3 can also be exemplified.

    The oligosaccharides in the present specification and the like include all those in which the number of monosaccharides constituting them is an integer of 2 to 20, but the oligosaccharides that can be identified in the present invention are those of the monosaccharides constituting these. The number is an even number and has a GalNAc residue at the reducing end.

    By the method of the present invention, it is possible to determine how CH, CSA or CSC of disaccharide units are linked in CH or CS whose sugar chain length and sulfate group position are unknown. It is also possible to identify a disaccharide unit (often referred to as CSE) in which both C-4 and C-6 positions of GalNAc contained in the CS sequence are sulfated.

Mass spectrometry in the present invention can be performed by an existing mass spectrometer capable of outputting general mass analysis data. Examples of the mass spectrometer that can be used in the present invention include Esquire, HCT (manufactured by Bruker Daltonics), LCQ, LTQ (manufactured by Thermo Fisher Scientific), and the like. In this specification and the like, mass spectrometry data includes both MS spectrum and MS ² spectrum data. In the present invention, mass spectrometry (MS) means that a sample to be analyzed is mass spectrometry in order to obtain mass spectrometry data. It means to call. The subjected to MS ² in the present invention, in order to obtain data of MS ² spectra refers to subjecting the test sample to the MS ² (2-stage mass spectrometry).

In the present invention, the MS spectrum refers to a spectrum reflecting the molecular weight of a sugar chain obtained by ionizing the sugar chain to be analyzed. In the present invention, the MS ² spectrum refers to various ions produced by applying energy in an apparatus to any ion (ionized sugar chain molecule to be analyzed) measured in the MS spectrum and breaking the ion due to excess energy. A spectrum that reflects the mass of a fragment (hereinafter also referred to as a fragment ion or a product ion).

    In the present invention, the m / z value is also referred to as a mass-to-charge ratio and refers to a value obtained by dividing the mass (m) of an ion by the number of charges (z) of the ion. The mass spectrometer reads the difference in ion motion according to the value of the mass-to-charge ratio in an electric field or magnetic field, and can calculate the mass of the ion by multiplying the m / z value by the valence of the ion.

    In the present invention, the “sugar chain length and sulfate group position” of CH or CS refers to the number of disaccharide units constituting CH or CS and the position at which a sulfate group is present in CH or CS. The basic skeleton of CH and CS is a repeating structure of a disaccharide unit consisting of GalNAc and GlcA. In the present invention, a structure having a sulfate group at the C-4 position and / or C-6 position of GalNAc is particularly handled. . Therefore, in the present invention, “setting the sugar chain length and sulfate group position of CH or CS consisting of disaccharide units” means setting the number of disaccharide units in the case of CH, and in the case of CS. Means setting a sulfate group position in GalNAc at an arbitrary position (including setting so as not to include a sulfate group) and setting the number of disaccharide units. In the present invention, “identify sugar chain length and sulfate group position” means an isomer based on the difference in the number of disaccharide units of CH or CS and the sulfate group position of GalNAc (including those containing no sulfate group). To clarify the structure.

    In the present invention, the “valence of fragment ions” refers to the number of deprotonations in fragment ions. In mass spectrometry, in CH, all or part of the carboxyl group in the molecule is usually deprotonated and ionized, and in CS, all or part of the carboxyl group and sulfate group in the molecule is usually deprotonated. To be ionized. Therefore, the valence of the fragment ion does not exceed the sum of the number of carboxyl groups and sulfate groups that can exist in the fragment ion.

In the method for identifying a sugar chain length and a sulfate group position of the present invention, m / z values substantially coincide with each other when m / z values of all fragment ions observed in an MS ² spectrum of CH or CS whose molecular structure is unknown. When CH or CS with the sugar chain length and sulfate group position set in the measured value of z value is attached to MS ² , all the fragment ions that can be generated by cleavage of the glycosidic bond in the disaccharide unit In addition to the fact that the predicted value of m / z value is included, this also means the case where only a part of the predicted value is included in the measured value. Here, when CH or CS is attached to MS ² , fragment ions generated by cleaving a portion other than the glycosidic bond in the disaccharide unit are included, so the number of the actual measurement values is the number of the prediction values. There is no less.

    A case will be described below where it is determined that the m / z values substantially match when only a part of the predicted value is included in the actual measurement value.

    If all fragments generated by cleavage of the glycosidic bond in the disaccharide unit are ionized, both B-type and Y-type ions, or both C-type and Z-type ions are generated (FIG. 2). In reality, however, one of the B-type and Y-type ions, or one of the C-type and Z-type ions may lose charge, and the lost fragment is not observed on the spectrum. Therefore, even if there exists an m / z value that is not included in the actual measurement value among the predicted values, the other fragment ion generated by the cleavage of the glycosidic bond for generating a fragment ion that gives the m / z value ( When the fragment ion giving the m / z value is B (Y) -type, the other fragment ion is Y (B) -type ion, and the fragment ion giving the m / z value is C (Z)- In the case of type, if the predicted value of the other fragment ion is Z (C) -type ion) is included in the actual measurement value, it is determined that the m / z values substantially match. Can do.

    When the predicted value and the actually measured value substantially coincide with each other, the sugar chain length and sulfate group position (unknown sugar chain length and sulfate group position) of the CH or CS that gave the actually measured value are The value is identified to be the same as the CH or CS sugar chain length and sulfate group position.

M / z values of all fragment ions that can be generated when a glycosidic bond in a disaccharide unit is cleaved for each of a plurality of CH or CS composed of disaccharide units set at different sugar chain lengths and sulfate groups. If a predicted value is calculated and a database is prepared in advance, an actual value of m / z value in an MS ² spectrum of CH or CS whose molecular structure is unknown is collated with the database, thereby obtaining the molecular structure. Unknown CH or CS sugar chain length and sulfate group position can be identified. In this case, the sugar chain length and sulfate group position of CH or CS that gave the predicted value of m / z value that substantially matches the actually measured value of the m / z value are the same as those of CH or CS whose molecular structure is unknown. It can be identified as the sugar chain length and the sulfate group position.

    Hereinafter, the regularity of the cleavage (cleavage) of glycosidic bonds of CH and CS oligosaccharide chains observed as described above in the present invention will be described.

    Here, it is assumed that the model of CH or CS has a GalNAc residue at the reducing end (right end side in FIG. 1) and a GlcA residue at the non-reducing end (left end side).

This sugar chain is a repetition of disaccharide units with a glucuronide bond in between, and is counted as the U = 1, 2, 3,... Nth unit from the reducing end. That is, the even-numbered sugar chains shown in FIG. 1 are “2N sugars”.
An arbitrary disaccharide unit is expressed as U = u.

Based on the previously reported nomenclature of fragment ions (Glycoconjugate Journal 5, 397-409, 1988), each glycoside cleavage ion of interest in the present invention is named as shown in FIG. That is, when the cleavage of the glucuronide bond in the disaccharide unit occurs on the GlcA side from the oxygen atom of the bond, the fragment ions on the non-reducing terminal side and the reducing terminal side generated by the cleavage are respectively represented by B-type ions and Called Y-type ion, the cleavage of the glucuronide bond in the disaccharide unit is more Ga than the oxygen atom of the bond.
When it occurs on the lNAc side, the non-reducing end side and reducing end side fragment ions generated by the cleavage are referred to as C-type ion and Z-type ion, respectively.

Here, B, C, Y and Z-type ions when U = u (represented as B _{2 (Nu) +1} , C _{2 (Nu) +1} , Y _2u-1 and Z _2u-1 ions, respectively) The signal m / z value (horizontal axis value) in the MS ² spectrum is given by the following equation.

1) B _{2 (Nu) +1} ion (m / z) = {[379 (N−u) +176] +80 sa−a} / a2) C _{2 (Nu) +1} ion (m / z) = {[379 (N−u) +176] + 18 + 80s−a} / a
3) Y _2u-1 ion (m / z) = {[379 (u-1) +203] + 18 + 80s-a} / a
4) Z _2u-1 ion (m / z) = {[379 (u-1) +203] + 80s-a} / a

N: Number of disaccharide units of the test sugar chain U: Number of disaccharide units from the reducing end to which the target glycosidic bond belongs 379: Mass of disaccharide units having no sulfate group 176: GlcA having no sulfate group Residue mass 203: Mass of GalNAc residue having no sulfate group 80: Mass per sulfate group 18: Mass of oxygen of glycoside bond s: Number of sulfate group contained in ion generated by cleavage of glycoside bond ( In many cases, it can be read from the MS ² spectrum)
a: Valence of ion generated by breaking glycosidic bond (in many cases, readable from isotope composition in MS ² spectrum)

Moreover, the regularity of fragmentation of CH and CS found in the present invention is as shown in FIG.
That is, when there is no sulfate group in the GalNAc residue present in the disaccharide unit from which the glycosidic bond is cleaved, cleavage that generates both or one of C _{2 (Nu) +1} and Z _2u-1 ions is performed. When a sulfate group is present at the C-4 position of a GalNAc residue that is present in the disaccharide unit in which the glycosidic bond is cleaved, both B _{2 (Nu) +1} and Y _2u-1 ions or If there is a sulfate group at the C-6 position of the GalNAc residue in the disaccharide unit that results in cleavage that results in cleavage of the glycosidic bond, B _{2 (Nu) +1} ion and Y _{2u − A} cleavage that produces both or _{one of the} ions and a cleavage that produces both or one of the C _{2 (Nu) +1} and Z _2u-1 ions.

In addition, when a sulfate group exists in both the C-4 position and the C-6 position of the GalNAc residue present in the disaccharide unit where the glycosidic bond is cleaved due to the fragmentation mechanism, B _{2 (Nu)} Cutting will occur where both ₊₁ ions and / or Y _2u-1 ions occur.

Here, the expression “both or one” is because fragments having no charge after the glycosidic bond cleavage in MS ² are not observed on the principle of the apparatus. In the case of the present invention, the sulfate group or carboxyl group is deprotonated upon ionization and acquires a negative charge and is detected, but not all such functional groups are deprotonated. Fragments that do not exist are not detected. That is, if any of the B-type ion and the Y-type ion, or any of the C-type ion and the Z-type ion is observed, the bond can be regarded as broken.

The method for predicting the m / z value of fragment ions in the MS ² spectrum of the present invention includes a method for predicting m / z values of all fragment ions that can occur in MS ² and m / z values of some fragment ions of interest. and predicting only the z value.

When predicting the m / z values of all fragment ions that can occur in MS ² , B = 1 is selected according to the presence of sulfate groups and position of sulfate groups of GalNAc in disaccharide units, , C, Y and Z-type ions (ie, when there is no sulfate group in GalNAc of the disaccharide unit to which the glycosidic bond to be cleaved belongs, C and Z-type ions, when there is a sulfate group at the C-4 position) Calculates the m / z value of B and Y-type ions, and B, C, Y and Z-type ions when a sulfate group exists only at the C-6 position.

On the other hand, it is also possible to predict only the m / z value of the fragment ion produced by the cleavage of a specific glycosidic bond. For example, if the fragment ion m / z value you want to know is only the B-type ion resulting from the cleavage of the glycosidic bond at U = u,
B _{2 (Nu) +1} ion (m / z) = {[379 _(Nu) +176] +80 sa−a} / a
Only the value of can be calculated.

    Subsequently, a method for identifying the sugar chain length and sulfate group position of CS using the method of the present invention using a model case will be described below with an example.

    When a MS spectrum of a certain CS oligosaccharide chain was obtained, a strong signal appeared at an m / z value of 467 as shown in FIG. When the spectrum was expanded in the horizontal direction (not shown), an isotope signal appeared at m / z values of 467.5 and 468.0, so this was a divalent ion, and the molecular mass was 467 × 2 + 2 (H) = 936 was calculated.

This molecular weight is usually 203 (GalNAc) × 2 + 176 (GlcA) × 2 + 18 (saturated oligosaccharide) +80 (sulfate group) according to a glycan composition program, a Web resource, or an empirical rule used by mass spectrometry researchers of glycans. X2 = 936, which is considered to be a CS4 sugar having a sulfate group bonded to two sites. When an MS ² spectrum was obtained for an ion having an m / z value of 467, it was as shown in FIG.

    In the case of tetrasaccharides, there are two glycosidic bonds to be noted according to the present invention as shown in FIG.

Here, ions giving both m / z values of 282 and 300 have already been observed on the MS ² spectrum. This is not GalNAc having one sulfate group, that is, other than Y ₁ ion and Z ₁ , and due to the appearance of both, the reducing terminal GalNAc is sulfated at the C-6 position due to fragmentation rules. .

By the way, if B3 and C3 ions that originate from the same cutting position are applied to the equation,
B _{2 (2-1) +1 (= 3)} ions (m / z) = {[379 (2-1) +176] + 80 × 1-1} / 1
= 634, and C _{2 (2-1) +1 (= 3)} ions (m / z) = {[379 (2-1) +176] + 18 + 80 × 1-1} / 1
= 652
Since both ions are observed on the MS ² spectrum, it can be confirmed that the C-6 position of the reducing terminal GalNAc is sulfated again.

Next, the position of one remaining sulfate group will be examined. Predicting the m / z values of Y ₃ and Z ₃ ions in the calculation formula yields the following.
Y _{2 × 2-1 (= 3)} ion (m / z) = {[379 (2-1) +203] + 18 + 80 × 1-1} / 1
= 679, and Z _{2 × 2-1 (= 3)} ions (m / z) = {[379 (2-1) +203] + 80 × 1-1} / 1 = 661.

However, these ions having m / z values have not been confirmed on the MS ² spectrum. As a precaution, the calculated value assuming divalent ions (a = 2) was also examined, but none of the ions could be confirmed in the same manner. On the other hand, although it is a weak signal,
B ₁ (m / z) = 175
C ₁ (m / z) = 193
Since the ions corresponding to each of these were confirmed on the MS ² spectrum, it is strongly suggested that the C-6 position of GalNAc involved in the glycosidic bond is sulfated.

As a result of the above examination, the sugar chain sample giving the MS spectrum and the MS ² spectrum of FIGS.
GlcA-GalNAc6S-GlcA-GalNAc6S
Can be determined.

    In the above model case, the case where all sulfate groups of GalNAc are located at the 6-position has been described. In this case, since all the B, C, Y and Z-type fragment ions can be generated, it can be said that the analysis is more complicated than those having other sulfate group positions. Therefore, in the case where the sulfate group is located at the 4-position, the case where both the 4-position and the 6-position are located, or the case where the sulfate group does not exist in all or part of the GalNAc, the sugar chain length is similarly applied. It is obvious to those skilled in the art that the sulfate group position can be identified. Moreover, if CH or CS has a sugar chain length of up to about 10 sugars, it is possible to predict the number of sulfate groups and sugar chain length from MS spectrum data by the sugar chain composition program, Web resources, or empirical rules. It will be apparent to those skilled in the art.

    Hereinafter, the present invention will be described in more detail with reference to examples. However, the scope of the present invention is not limited to the examples.

[Example 1]
Analysis of CH Fragmentation Rules in MS ² CH oligosaccharides without sulfate groups were analyzed to examine their regularity of fragmentation (fragmentation) by MS ⁿ .

(1) Preparation of CH oligosaccharides All CH oligosaccharides were obtained from the Institute of Molecular Medical Science, Aichi Medical University. A simple procedure is as follows. Saturated tetrasaccharide and hexasaccharide (CH4 and CH6) of CH are described in J. Am. Biol Chem. 277, 21567-21575 (2002) and Anal. Biochem. 365, 62-73 (2007). That is, CH (manufactured by Seikagaku Corporation) is digested with testicular hyaluronidase (type-5, manufactured by Sigma), and the resulting oligosaccharides are separated by anion exchange column chromatography and then desalted by size exclusion chromatography. did. CH3 and CH5 having GalNAc at the non-reducing end were obtained by treating CH4 and CH6 with β-glucuronidase (derived from bovine liver, type B-1, manufactured by Sigma). Oligosaccharides longer than CH6 were synthesized enzymatically by CH polymerase using CH6 as the first glycosylation acceptor.

(2) ESI-MS ⁿ analysis conditions For negative ion mode ESI-IT MS, Esquire 3000 Plus (manufactured by Bruker Daltonik GmbH) was used. The oligosaccharide was dissolved in 50% methanol (pH 6.0) containing 2.5 mM ammonium acetate, and directly injected into the apparatus using a syringe pump at a flow rate of 360 μL / hour. The operation was performed by setting a capillary voltage of −3.8 kV, an end-plate offset of −500 V, a dry nitrogen gas flow rate of 4.0 L / min, and a drying temperature of 300 ° C. MS ⁿ data by the CID method was obtained by adjusting the fragmentation energy to 1.0V. The scan molecular weight range was set to m / z 50-1000 or 1500, and the scan resolution was set to 5500 Da / s.

(3) Results and Discussion (Signal attribution is Glycoconjugate Journal)
5, 397-409, 1988)
Negative ion ESI mass spectra were obtained for CH oligosaccharides of trisaccharide (CH3) to octasaccharide (CH8) (Table 1). In CH4 to CH8, [M-2H] ^2−, which is a double deprotonated molecule, remarkably appeared, while in CH3, [M−H] ⁻ appeared remarkably. Subsequently, CID MS ⁿ experiments were performed to examine their fragmentation characteristics.

FIG. 7 shows an example of a mass spectrum of MS ⁿ product ions ((A): CH5, (
B): CH6). Almost all observed product ions (fragment ions) originated from glycosidic cleavage and found common fragmentation rules. That is, saturated CH oligosaccharides lost monosaccharide residues every single CID process, resulting in C-type ions.
Three mechanisms can be proposed for these fragmentations that mainly produce C-type ions (FIG. 8). The elimination of GalNAc residues can occur by charge-inducing fragmentation (FIGS. 8A and 8C). On the other hand, a mechanism for forming an oxirane ring is also conceivable for elimination of the GlcA residue (FIG. 8B).

[Example 2]
Analysis of CS fragmentation rules in MS ² Disaccharide, tetrasaccharide and hexasaccharide CSA and CSC oligosaccharides having a uniform sequence were analyzed by MS ⁿ . Fragmentation features unique to sulfated sites were observed in the MS ² spectrum. Based on the fragmentation results, the possible cleavage mechanism is also discussed.
(1) Preparation of CS oligosaccharide All CS oligosaccharides were prepared by Seikagaku Corporation (FIG. 9). CSA oligosaccharides (CSA2, CSA4 and CSA6) having a uniform sequence of disaccharide, tetrasaccharide and hexasaccharide were prepared from CSA (manufactured by Seikagaku Corporation) isolated from sturgeon notochord. This CSA is much more abundant than that isolated from whale cartilage, with disaccharide units having GalNAc with sulfated C-4. This CSA is digested with testicular hyaluronidase (type V, purchased from Sigma), and the resulting oligosaccharide can be separated into an NH ₂ -modified silica column (YMC-Pack NH2, which can separate isomeric oligosaccharides having different sulfation sites. It was separated repeatedly by anion exchange HPLC connected to YMC) and then desalted by size exclusion chromatography. The structure of the oligosaccharide thus obtained was analyzed by both ¹ H-NMR measurement and enzyme digestion analysis, and it was confirmed that the C-4 site had only sulfated GalNAc.

    Uniform CSC disaccharide, tetrasaccharide and hexasaccharide oligosaccharides (CSC2, CSC4 and CSC6) in which only the C-6 site of GalNAc is all sulfated are commercially available non-sulfated chondroitin ( CH, manufactured by Seikagaku Corporation). Sulfation with primary hydroxyl group (C-6 position of GalNAc) mainly sulfated by sulfation of CH with pyridine-sulfur trioxide complex in N, N-dimethylformamide at low temperature (0 ° C) CH was obtained. Thereafter, oligosaccharides produced by digestion with testicular hyaluronidase were repeatedly separated by size exclusion chromatography to obtain the desired oligosaccharides. The structure of the oligosaccharide thus obtained was analyzed by enzymatic digestion, and it was confirmed that all GalNAc contained in CS was sulfated only at the C-6 position.

(2) ESI-MS ⁿ analysis conditions For the negative ion mode ESI-IT MS, Esquire 3000 Plus (manufactured by Bruker Daltonik GmbH) was used. The oligosaccharide sample was dissolved in methanol (spectrophotometric grade, sold by Aldrich Chemical) containing 2.5 mM ammonium acetate, and a nanospray device (PicoTip ^™ Emitter, manufactured by New Objective) was used at a flow rate of 20 μL / h. Injected into the device. The operation was performed by setting a capillary voltage of −2.5 kV, an end plate offset of −500 V, a dry nitrogen gas flow rate of 5.0 L / min, and a drying temperature of 300 ° C. CID-MS ² data was obtained by adjusting the fragmentation energy to 1.0V. The scan mass range was m / z 50-1500, and the scan resolution was 5500 m / z / s.

(3) Results and discussion 1) CSA oligosaccharide with uniform sequence CSA derived from sturgeon notochord has a high content (> 90%) of GlcA-GalNAc4S sequence. Suitable for the preparation of oligosaccharides. The oligosaccharides obtained from this CSA, namely CSA2, CSA4 and CSA6, were subjected to MS ⁿ .

Each MS spectrum of the CSA oligosaccharide shows a simple single signal corresponding to [M-nH] ^n- (where "n" is equal to the number of sulfate groups in the molecule) under the conditions employed in this study. (Data omitted). That is, CSA2, CSA4, and CSA6 have [M−H] ⁻ (m / z 476), [M−2H] ²⁻ (m / z 467) and [M−3H] ³⁻ (m / z) in the MS, respectively. 464) ions. Subsequently, each ion was subjected to MS ⁿ as a precursor ion. Figure 10 shows the product ion mass spectrum of oligosaccharides in MS ^2. They showed a highly regular glycosidic cleavage mode and produced B-type and Y-type product ions without desulfation. All of them could be attributed under the assumption that each negative charge is located in each sulfate group (Table 2). In fragmentation of non-sulfated CH oligosaccharides, most of the fragments are C-type ions, and the negative charge is considered to be located at the carboxyl group. Therefore, it was demonstrated that sulfation of the C-4 hydroxyl group of GalNAc has an important influence on the fragmentation pattern of oligosaccharides.

2) CSC oligosaccharide with uniform sequence Similarly, CSC oligosaccharide with uniform sequence was analyzed. Their MS ² spectra are shown in FIG. 11 and showed very complex features as opposed to the uniform sequence of CSA oligosaccharides. All of the product ions observed with CSA oligosaccharides are also observed with CSC oligosaccharide fragments, and it is particularly noteworthy that B-type and Y-type ions are in common (FIG. 10). In addition, in the same glycosidic bond, approximately equal amounts of C-type and Z-type ions were produced, comparable to B-type and Y-type ions. Interestingly, some product ions have lost sulfate groups to form hydroxyl groups (eg, [M-SO ₃ -nH] ^n- , B ₃ -SO ₃ , C ₃ -SO ₃ etc.) It attracted attention. The desulfated species and C-type and Z-type ions were not detected at all as trace fragments of the CSA oligosaccharide having a uniform sequence, or even if detected. That is, it was found that the precursor ion of CSC oligosaccharide causes various fragmentation.

3) Fragmentation mechanism The data obtained in this study have shown that the fragmentation (fragmentation) pattern of the CS backbone clearly reflects the sulfation position in the GalNAc residue. These fragmentation mechanisms can be considered as follows.

I) Glycoside cleavage producing B-type and Y-type ions on the reducing end side of GlcA This cleavage occurred in the fragmentation of both CSA and CSC oligosaccharides, but not in CH oligosaccharides. That is, this is considered as a unique cleavage process for the sulfated sequence of CS, and is understood by a mechanism involving the C-2 hydroxyl group of GlcA (FIGS. 12 (B) and (C), Non-Patent Document 5). . This process is fragmentation independent of charge position (charge remote fragmentation) and thus occurs independently of the position of the sulfate group in the GalNAc residue.

II) Glycoside cleavage producing C-type and Z-type ions on the reducing end side of GlcA This type of glucuronide cleavage was observed in CSC oligosaccharides but not in CSA. On the other hand, it is a cleavage pattern that occurs even with non-sulfated CH oligosaccharides (FIG. 12A). In fragmentation of CH oligosaccharides by charge induced fragmentation, the negative charge present on the carboxyl group triggers the cleavage process giving C-type and Z-type ions (FIG. 8). However, in the general case of sugars that are originally sulfated, they undergo fragmentation (charge remote fragmentation) that is not affected by charge (Non-patent Document 5 and Rapid Commun. Mass Spectrom. 19 1788-1796 (2005)). Here, regarding the fragmentation of CSC oligosaccharide, it is possible to understand the fragmentation mechanism by assuming proton transfer (FIG. 12C).

As described above, fragmentation of CH and CS oligosaccharides with ESI-MS ² could be understood based on the cleavage mechanism specific to each structure. These organized findings could be extended not only to the distinction of both CSA and CSC oligosaccharides, but also to the sequencing of various CS-related oligosaccharides.

More Universal CS Sequencing Protocol Next, it will be described that the CS can be predicted for any sequence based on the regularity of the MS ² spectrum.

In the above examples, standard oligosaccharides with a uniform sequence of CH, CSA and CSC were analyzed. Based on the obtained data, it was possible to find a fragmentation rule specific to the sulfated position of GalNAc (FIG. 12 shows the fragmentation mechanism). By combining these results, it can be understood that the fragmentation behavior of a CS oligosaccharide having a heterogeneous sulfated sequence can also be estimated as shown in FIG.

    The present invention provides a means for identifying the CH and CS sugar chain lengths and sulfate group positions using a multi-stage mass spectrometer, whereby the CH and CS sugar chain lengths and sulfate group positions and functions in the living body are provided. Therefore, it is possible to contribute to diagnosis and treatment of various diseases caused by an abnormality in the chemical structure of CH or CS.

It is the figure which showed the molecular structure model of CH or CS which has a GalNAc residue in a reducing end and a GlcA residue in a non-reducing end. It is the figure which showed the nomenclature of the fragment ion produced by the cutting | disconnection of a glycosidic bond. It is a figure which shows the relationship between the sulfate group position of GalNAc residue, and the type of the fragment ion produced by the cutting | disconnection of the glycosidic bond in the disaccharide unit to which this GalNAc residue belongs. It is a figure which shows the MS spectrum of a model case. It is a diagram illustrating a MS ² spectra model case. It is the figure which showed the glycoside bond which should be noted in the model case, and the type of the fragment ion which can arise from there. FIG. 2 shows typical ESI-MS ⁿ fragments of CH oligosaccharides ((A): CH5, (B) CH6). It is a figure which shows the proposed mechanism of MS ⁿ fragmentation of saturated CH oligosaccharide ((A): Cleavage mechanism of the glucuronide bond which produces a C-type ion, (B): C or Z-type ion (C): Glucuronide bond cleavage mechanism producing C-type ions). It is a figure which shows the structure ((A): CSA oligosaccharide of a uniform arrangement | sequence, (B): CSC oligosaccharide of a uniform arrangement | sequence) analyzed in the present Example. Is a diagram showing an ESI-MS ² spectra of CSA oligosaccharides analyzed uniform sequence ((A): CSA2, ( B) CSA4, (C) CSA6). The fragmentation pattern of each oligosaccharide is also illustrated ((D): CSA2, (E): CSA4, (F): CSA6). Is a diagram showing an ESI-MS ² spectra of CSC oligosaccharides analyzed uniform sequence ((A): CSC2, ( B) CSC4, (C): CSC6). The fragmentation pattern of each oligosaccharide is also illustrated ((D): CSC2, (E): CSC4, (F): CSC6). It is the figure which showed the presumed cleavage mechanism of the glycosidic bond of CH oligosaccharide ((A) in a figure), CSA oligosaccharide ((B) in a figure), and CSC oligosaccharide ((C) in a figure). It is the figure which showed the cutting pattern in CS of a uniform arrangement | sequence and a non-uniform arrangement | sequence.

Claims (5)

A method for predicting an m / z value in an MS ² spectrum of a fragment ion generated when chondroitin or chondroitin sulfate is subjected to MS ² , comprising at least the following steps:
(I) a step of setting a sugar chain length and a sulfate group position of chondroitin or chondroitin sulfate consisting of a disaccharide unit that is a target of prediction of m / z value in MS ² spectrum;
(II) a step of selecting a disaccharide unit to which a glycosidic bond cleaved when the chondroitin or chondroitin sulfate of (I) is subjected to MS ² ;
(III) Steps for determining the following numerical values of (i) to (iv) for chondroitin or chondroitin sulfate of (I); and (i) N: number of disaccharide units,
(Ii) u: the number of the disaccharide unit selected in the above (II) when the disaccharide unit is numbered as one unit in order from the reducing end,
(Iii) s: the number of sulfate groups of the fragment ion generated by cleavage of the glycosidic bond belonging to the disaccharide unit selected in (II) above,
(Iv) a: the valence of the fragment ion generated by the cleavage of the glycosidic bond belonging to the disaccharide unit selected in (II) above,
(IV) Each numerical value obtained in the step (III) was selected in the above (II) by applying to the following formulas (A) to (D) according to any of the following (A) to (C): Determining an m / z value in the MS ² spectrum of a fragment ion selected from B, C, Y and Z-types that can be generated by cleavage of a glycosidic bond belonging to a disaccharide unit;
(A) When N-acetylgalactosamine constituting the disaccharide unit to which the glycosidic bond selected in (II) above does not have a sulfate group, the following formulas (b) and (d):
(B) When it has a sulfate group at the C-4 position of the N-acetylgalactosamine, the following formulas (a) and (c), or (C) the C-6 position of the N-acetylgalactosamine All of the following formulas (a) to (d)
(A) B _{2 (Nu) +1} ion (m / z) = {[379 (N−u) +176] +80 sa−a} / a,
(B) C _{2 (Nu) +1} ion (m / z) = {[379 (N−u) +176] + 18 + 80s−a} / a,
(C) Y _2u-1 ion (m / z) = {[379 (u-1) +203] + 18 + 80s-a} / a,
(D) Z _2u-1 ion (m / z) = {[379 (u-1) +203] + 80s-a} / a. A method for identifying the sugar chain length and sulfate group position of chondroitin or chondroitin sulfate with unknown molecular structure, comprising at least the following steps;
(V) A step of attaching chondroitin or chondroitin sulfate of unknown molecular structure to MS ² to obtain m / z values of all fragment ions observed in the obtained MS ² spectrum,
(VI) All fragments that can be produced by cleaving chondroitin or chondroitin sulfate in which the sugar chain length and sulfate group position are set to MS ² by cleaving the glycosidic bond in the disaccharide unit by the method according to claim 1 A step of obtaining predicted values of m / z values in the MS ² spectrum for ions, and (VII) all m / z values obtained by the step (V) and all obtained by the step (VI). When the molecular structure is unknown, the sugar chain length and sulfate group position of chondroitin or chondroitin sulfate whose molecular structure is unknown are compared in the step (VI). Identifying the set sugar chain length and sulfate group position.   (VI) and (VII) until all m / z values acquired by the step (V) and the predicted values of all m / z values acquired by the step (VI) substantially match. The identification method according to claim 2, further comprising a step of repeating the step of). A method for identifying the sugar chain length and sulfate group position of chondroitin or chondroitin sulfate with unknown molecular structure, comprising at least the following steps;
(VIII) When each of a plurality of chondroitin or chondroitin sulfates composed of disaccharide units set at different sugar chain lengths and sulfate group positions by the method according to claim 1 is subjected to MS ² , The predicted value of m / z value in the MS ² spectrum is obtained for all fragment ions that can be generated by the cleavage of the glycosidic bond, and the correspondence between the sugar chain length and sulfate group position and the predicted value of m / z value Creating a database about
(IX) A step of attaching chondroitin or chondroitin sulfate of unknown molecular structure to MS ² to obtain m / z values of all fragment ions observed in the obtained MS ² spectrum, and (X) IX) All m / z values acquired in step (IX) are compared with the m / z values in the database created in step (VIII), and all m / z values acquired in step (IX). And finding the sugar chain length and sulfate group position of chondroitin or chondroitin sulfate having a predicted value of m / z value that substantially coincides with.   The method according to claim 1, wherein the chondroitin or chondroitin sulfate is chondroitin oligosaccharide or chondroitin sulfate oligosaccharide, respectively.

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