JP4569236B2 - Method for derivatizing protein or peptide with sulfonic acid, and method for determining amino acid sequence of protein or peptide - Google Patents

Description

  The present invention relates to the field of determining the amino acid sequence of a protein or peptide in a mass spectrometer.

  Conventionally, as a method of derivatizing a protein or peptide with a sulfonic acid, a compound having a sulfonic acid or a sulfonyl group is directly reacted with the N-terminus of the protein or peptide. However, in such a method, since the reactive species has a strongly acidic group, there is a problem in the reaction efficiency to the N-terminus of the protein or peptide.

  In addition, Proc. Natl. Acad. Sci. USA Vol 96, PP7131-7136, June 1999 includes a sulfonic acid at the N-terminus of a peptide using a reagent such as 2-sulfobenzoic acid cyclic acid anhydride or chlorosulfonylacetyl chloride. Methods for introducing groups are described.

  On the other hand, in Rapid Commun. Mass Spectrom. 12, 603-608 (1998), by modifying a compound containing a Br atom at the N-terminus of a peptide, the characteristic 2 mass unit difference of the Br atom is obtained from MS data. A method for identifying an object based on a doublet peak is known.

Proceeding of the National Academy of Science of the United States of America, Vol. 96, June 1999 , P. 7131-7136 Rapid Communications in Mass Spectrometry, Vol. 12, 1998, p. 603-608

  However, in the method described in Proc. Natl. Acad. Sci. USA Vol 96, PP7131-7136, June 1999, the above-mentioned reagent is allowed to act on the peptide fragment produced by enzymatic digestion. It cannot be selectively recovered, and only the N-terminal peptide fragment cannot be sulfonated. Further, the N-terminal peptide fragment cannot be selectively recovered even by the method described in Rapid Commun. Mass Spectrom. 12, 603-608 (1998).

  Accordingly, a first object of the present invention is to provide a method for derivatizing a sulfonic acid by efficiently introducing a sulfonic acid group into the N-terminus of a protein or peptide. For this purpose, a sulfonic acid derivatization can be performed while selectively recovering an N-terminal fragment of a protein or peptide, and a method useful for mass spectrometry is provided.

  The second object of the present invention is to provide a method capable of simply and efficiently analyzing a protein or peptide by mass spectrometry. For this purpose, it is possible to efficiently analyze the N-terminal peptide fragments of proteins or peptides by selectively recovering them and easily distinguishing the recovered N-terminal peptide fragments from other contaminants in the mass spectrum. Provide a way to do it.

  A third object of the present invention is to provide a compound that can be used for N-terminal modification of a protein or peptide. For this purpose, the N-terminus of a protein or peptide is efficiently sulfonated, and a compound useful for selective recovery and mass spectrometry of an N-terminal peptide fragment is provided.

  A fourth object of the present invention is to provide an intermediate for efficiently derivatizing the N-terminus of a protein or peptide with a sulfonic acid. For this purpose, intermediates useful for selective recovery and mass spectrometry of N-terminal peptide fragments are provided.

1. The following inventions (1) to ( 12 ) relate to a method of derivatizing a protein or peptide with a sulfonic acid.

(1) below shows a modification step with compound A, have rows and cleavage step, after the modification step, prior to the cleavage step, the fragmentation step and the separation step the line cormorants form.
(1) A protein in which the N-terminus is modified with the compound A by reacting a compound A containing an atom showing a characteristic isotope composition in mass spectrometry and a disulfide group with the N-terminus of the protein or peptide Or a modification step to obtain a peptide;
In the modified protein or peptide, by converting to sulfonic acid groups by cleaving the disulfide bonds of the disulfide groups, see contains a cleavage step of sulfonic acid derivatives of the modified protein or peptide,
After the modification step and before the cleavage step,
By fragmenting the protein or peptide modified by the compound A, one N-terminal peptide fragment derived from the N-terminus of the protein or peptide and modified by the compound A, and other than the N-terminal peptide fragment A fragmentation step to obtain one or more other peptide fragments of
A separation step of selectively recovering the N-terminal peptide fragment by separating it from the other peptide fragments,
In the cleavage step, a protein or peptide that obtains a sulfonic acid derivatized N-terminal peptide fragment by cleaving the disulfide bond derived from the compound A in the N-terminal peptide fragment and converting it to a sulfonic acid group Method for derivatizing sulfonic acid.

  The following (2) to (4) show the structure of the compound A in (1).

(2) The method for derivatizing the protein or peptide according to (1), wherein the compound A has a functional group capable of reacting with the N-terminus of the protein or peptide.
(3) The functional group capable of reacting with the N-terminus of the protein or peptide is a carboxyl group, isothiocyanate group, succinimidyloxycarbonyl group, p-nitrophenoxycarbonyl group, pentafluorophenoxycarbonyl group, and tetra A method for derivatizing a protein or peptide according to (2) selected from the group consisting of a fluorosulfophenoxycarbonyl group.
(4) The method for derivatizing a protein or peptide according to any one of (1) to (3), wherein the atom having the isotopic composition is Br, Cl or B.

The following (5) and (6) show the cleavage step in (1).
(5) The protein or peptide according to any one of (1) to (4) is converted to a sulfonic acid derivatization by performing oxidative cleavage of the disulfide bond in the cleavage step. Method.
(6) The protein or peptide according to any one of (1) to (4), wherein the conversion to the sulfonic acid group in the cleavage step is performed by reductively cleaving the disulfide bond and then performing an oxidation reaction. To derivatize sulfonic acid.

The following (7) and (8) show a mode in which the protection step is performed before the modification step in (1).
(7) The method for derivatizing a protein or peptide according to any one of (1) to (6), further comprising a protecting step for protecting a side chain amino group of the protein or peptide before the modifying step. .
(8) The method of derivatizing the protein or peptide according to (7), wherein the side chain amino group is protected by guanidinolation in the protection step.

The following ( 9 ) and ( 10 ) show forms using the immobilized compound A.
( 9 ) In the modification step, first, an immobilized compound A in which a solid support is further bound to the compound A is prepared, and then the immobilized compound A is reacted with the N-terminus of the protein or peptide. The protein or peptide in any one of (1)-( 8 ) which obtains the protein or peptide by which the said N terminal was modified by the said fixed type compound A by making it sulfonate.
( 10 ) In the modification step, first, an immobilized compound A in which a solid support is further bound to the compound A is prepared, and then the immobilized compound A is reacted with the N-terminus of the protein or peptide. To obtain a protein or peptide whose N-terminus is modified by the immobilized compound A,
In the fragmentation step, an N-terminal peptide fragment modified with the immobilized compound A and the other peptide fragment are obtained,
In the separation step, the N-terminal peptide is selectively recovered by eluting the other peptide fragments,
In the cleavage step, the N-terminal peptide fragment derivatized with sulfonic acid is obtained by cleaving the disulfide bond of the N-terminal peptide fragment, wherein the protein or peptide according to any one of (1) to ( 8 ) is sulfone Method of acid derivatization.

The following ( 11 ) and ( 12 ) show forms using the biotinylated compound A.
( 11 ) In the modification step, first, a biotinylated compound A in which a biotinyl group is further bonded to the compound A is prepared, and then the biotinylated compound A is reacted with the N-terminus of a protein or peptide. To obtain a protein or peptide whose N-terminus is modified with the biotinylated compound A, wherein the protein or peptide according to any one of (1) to ( 8 ) is derivatized with sulfonic acid.
( 12 ) In the modification step, first, a biotinylated compound A in which a biotinyl group is further bonded to the compound A is prepared, and then the biotinylated compound A is reacted with the N-terminus of a protein or peptide. To obtain a protein or peptide whose N-terminus is modified by the biotinylated compound A,
In the fragmentation step, an N-terminal peptide fragment modified with the biotinylated compound A and the other peptide fragment are obtained,
In the separation step, the N-terminal peptide fragment is selectively adsorbed on the support by adsorbing the N-terminal peptide fragment to a solid support on which avidin is immobilized and eluting the other peptide fragments. To collect
The protein or peptide according to any one of (1) to ( 8 ) , wherein in the cleavage step, a disulfide bond of the adsorbed N-terminal peptide fragment is cleaved to obtain a sulfonic acid-derivatized N-terminal peptide fragment. To derivatize sulfonic acid.

2. The following invention ( 13 ) relates to a method for determining the amino acid sequence of a protein or peptide.
( 13 ) Detecting a specific peak based on the isotopic composition of a sulfonic acid derivative of a protein or peptide obtained by using the method according to any one of (1) to ( 12 ) by mass spectrometry To determine the amino acid sequence of the protein or peptide.

3. The following inventions, relates to a compound A.
A compound having the atoms exhibiting a characteristic isotope composition in mass spectrometry, the disulfide group, and a functional group capable of reacting with the N-terminus of the protein or peptide A.
To sulfonic acid derivative of the protein or peptide, or used to determine the amino acid sequence of the protein or peptide by mass spectrometry, the compound A.
Compound A above , wherein the solid support is further bound.
Bi Ochiniru group is further bound, the compound A.

4). The following inventions relates modified proteins or peptides.
Characteristic and atomic showing the isotopic composition, based on the modified proteins or peptides in having a disulfide group in mass analysis.
And atoms with pre Symbol isotopic composition, the group having a disulfide group, further solid phase support is attached, said protein or peptide.
And atoms with pre Symbol isotopic composition, based on having a disulfide group, which is further linked to biotinyl group, the protein or peptide.

  According to the present invention, it is possible to provide a method for derivatizing a sulfonic acid by efficiently introducing a sulfonic acid group into the N-terminus of a protein or peptide. It is also possible to provide a method useful for mass spectrometry of a sulfonic acid derivatized protein or peptide that can be sulfonated while selectively recovering an N-terminal peptide fragment of the protein or peptide. it can.

  ADVANTAGE OF THE INVENTION According to this invention, the method which can analyze protein or a peptide simply and efficiently by mass spectrometry can be provided. In addition, a method that allows efficient analysis by selectively recovering N-terminal peptide fragments of proteins or peptides and easily distinguishing N-terminal peptide fragments recovered in mass spectra from other contaminants. Can be provided.

  According to the present invention, a compound that can be used for N-terminal modification of a protein or peptide can be provided. In addition, it is possible to efficiently derivatize a protein or peptide and to provide a compound useful for selective recovery and mass spectrometry of an N-terminal peptide fragment.

  According to the present invention, an intermediate for efficiently derivatizing a protein or peptide with a sulfonic acid can be provided. In addition, intermediates useful for selective recovery and mass spectrometry of N-terminal peptide fragments can be provided.

  First, the present invention is a method for derivatizing a sulfonic acid by introducing a sulfonic acid group into the N-terminus of a protein or peptide. The method of the present invention includes a modification step of modifying the N-terminus of a protein or peptide with a disulfide group-containing compound (described later as Compound A), and a cleavage step of cleaving the disulfide group to convert it to a sulfonic acid group. In some cases, a protection step is included before the modification step, and a fragmentation step and a separation step are included between the modification step and the cleavage step.

[Protection process]
In the method of the present invention, usually, prior to the modification step, a protection step for protecting the side chain amino group of the amino acid residue having a side chain amino group of the protein or peptide to be derivatized with sulfonic acid can be performed. . Examples of amino acid residues having a side chain amino group include lysine residues having an ε-amino group and ornithine residues having a δ-amino group. In the protection step, a method that selectively reacts only with the side chain amino group and does not react with the N-terminal amino group of the protein or peptide can be used without any particular limitation. For example, it is preferable to carry out a method of protecting an amino group with a guanidino group by a guanidinolation reaction. In the guanidinolation reaction, for example, a lysine residue is converted into a homoarginine residue, and an ornithine residue is converted into an arginine residue. As a result, a protein or peptide having a protected side chain amino group is obtained.

  An example in which a peptide model containing only lysine as an amino acid residue having a side chain amino group is protected by guanidinolation is shown, and this step is shown in the following formula. In the formula, a to l represent amino acid residues having no side chain amino group, Lys represents a lysine residue, and Hor represents a homoarginine residue.

As guanidino reaction is shown above formula, to convert the side chain amino group (-NH _2), the guanidino group _{(-NHC (= NH) NH 2} ). As the guanidinating reagent, O-methylurea, S-methylisothiourea, 1-guanylu 3,5-dimethylpyrazole and the like may be used. In practice, for example, a protein to be analyzed and a guanidinating reagent are mixed in a basic solution such as aqueous ammonia to carry out the reaction. In this way, a protein or peptide having a side chain amino group protected with a guanidino group is obtained.

  In the protection step, only the side chain amino group is selectively protected and the N-terminal amino group of the protein or peptide is not protected. Therefore, the N-terminal amino group can be selectively modified in the modification step.

[Modification process]
In the modification step, the N-terminal of the protein or peptide to be derivatized with sulfonic acid or the protected protein or peptide obtained by the above-described protection step (hereinafter simply referred to as protein or peptide) is used as the disulfide group-containing compound. (Denoted as compound A).

  Compound A in the method of the present invention is a compound having an atom having an isotopic composition characteristic in mass spectrometry, a functional group capable of reacting with the N-terminus of a protein or peptide, and a disulfide group. A schematic structure of Compound A is shown below. Here, a and b represent an organic group, X represents an atom having a characteristic isotope composition in mass spectrometry, and Y represents a functional group capable of reacting with the N-terminus of a protein or peptide.

  Furthermore, specific examples of Compound A are shown below. Here, although the immobilization type compound A and the biotinylated type compound A are mentioned, these details are mentioned later. In the immobilized compound A shown below, a circle represents a solid support.

In the present invention, an atom having an isotope composition ratio characteristic in mass spectrometry has an isotope, and at least two of the isotopes have a width corresponding to the mass difference in mass spectrometry. Atoms present in such an amount that they can be detected with an ionic strength that can be identified as a typical pair peak or multiple peak. Examples of such atoms include Br, Cl, and B. In particular, Br atom, and an ⁷⁹ Br and ⁸¹ Br isotopically, as the natural abundance, the abundance ratio of the abundance ratio of the high abundance ⁷⁹ Br and 100 ⁸¹ Br 97.2776 (i.e. ⁷⁹ Br: ⁸¹ Br = 50.69%: 49.31%). Therefore, for example, in the case of a protein or peptide modified by using Compound A containing one Br atom, two peaks of approximately the same intensity having a difference of 2 Da on the mass spectrum are detected as characteristic pair peaks. . From this viewpoint, in the present invention, it is particularly preferable to use the compound A containing a Br atom.

  In the present invention, since Compound A contains atoms having a characteristic isotope composition ratio, it is particularly useful when used for amino acid sequencing by mass spectrometry. That is, when a sulfonic acid derivative of a protein or peptide obtained by this method is subjected to analysis by mass spectrometry, it is contained in the sample in addition to the sulfonic acid derivative on the mass spectrum based on its characteristic spectral peak. There is an advantage that the impurities to be obtained can be identified while being distinguished.

  As the functional group capable of reacting with the N-terminus of the protein or peptide, a carboxyl group is usually selected. In addition, an active group such as an isothiocyanate group, an active ester group such as a succinimidyloxycarbonyl group, a p-nitrophenoxycarbonyl group, a pentafluorophenoxycarbonyl group, and a tetrafluorosulfophenoxycarbonyl group may be used. . Compound A may further have a sulfonic acid group. The sulfonic acid group may be bonded to a functional group capable of reacting with the N-terminus. The disulfide group constitutes a part of the main chain of Compound A.

  Compound A can be obtained, for example, by the following method. That is, a compound having a functional group capable of reacting with an amino group of an amino acid or an amino acid derivative and a disulfide group is prepared as a precursor of Compound A, and an amino acid or amino acid derivative containing the atom is bonded thereto. Can be obtained by: Examples of the precursor of Compound A include AEDP (3-([2-aminoethyl] dithio) propionate hydrochloride), cystine derivatives, and the like. Amino acids include α-, β-, γ-, and δ-amino acids. Furthermore, peptides or proteins are also included as amino acid derivatives (however, peptides or proteins as amino acid derivatives here are prepared separately from proteins or peptides to be analyzed or sulfenylated in the present invention). .

  Compound A can react such an amino acid or amino acid derivative with the precursor in the presence of a base using a condensing agent. Examples of the condensing agent include EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), dicyclohexylcarbodiimide, diisopropylcarbodiimide and the like. The amount of the condensing agent can be used, for example, 1 to 50 equivalents with respect to the amino acid or amino acid derivative. Examples of the base include triethylamine, trimethylamine, diisopropylpyrroleamine, N-ethylmorpholine and the like. The amount of the base can be used, for example, 1 to 50 equivalents with respect to the amino acid or amino acid derivative. The said reagent can be used individually or in combination of 2 or more types.

  Examples of the solvent used in this reaction include dimethylformamide, distilled water, dimethylacetamide, N-methylpyrrolidone and the like. These can be used alone or in combination of two or more. The organic solvent can be used at any concentration. Moreover, as reaction conditions, it can be made to react at 0 to 70 degreeC for 0.1 to 24 hours, for example. Those conditions can be appropriately determined by those skilled in the art.

  In this step, the protein or peptide and the compound A can be reacted using a condensing agent in the presence of a base. Examples of the condensing agent include EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), dicyclohexylcarbodiimide, diisopropylcarbodiimide and the like. The amount of the condensing agent can be used, for example, 1 to 50 equivalents with respect to the protein or peptide. Examples of the base include triethylamine, trimethylamine, diisopropylpyrroleamine, N-ethylmorpholine and the like. The amount of the base can be used, for example, 1 to 50 equivalents relative to the protein or peptide. The said reagent can be used individually or in combination of 2 or more types.

  Examples of the solvent used in this reaction include dimethylformamide, distilled water, dimethylacetamide, N-methylpyrrolidone and the like. These can be used alone or in combination of two or more. The organic solvent can be used at any concentration. Moreover, as reaction conditions, it can be made to react at 0 to 70 degreeC for 0.1 to 24 hours, for example. Those conditions can be appropriately determined by those skilled in the art.

  When compound A has an active group as described above as a functional group capable of reacting with the N-terminus of a protein or peptide, an aqueous buffer such as a phosphate buffer having a pH of 6.5 to 8.0 is used without using a condensing agent. The reaction can be carried out in the liquid at 0 to 70 ° C. for 0.1 to 24 hours.

  In the method of the present invention, a solid support further bound to compound A (referred to as immobilized compound A) may be used. As such a solid-phase support, a solid-phase support having a functional group capable of binding to a portion other than the reaction point with the N-terminus of the protein or peptide in the main chain of the compound A is used without particular limitation. be able to. For example, a resin having the above functional group can be used. When AEDP is used as the compound A, isothiocyanate glass beads can be used. At this time, the isothiocyanate glass beads and the AEDP can be immobilized by reacting them under basic conditions. In the immobilized compound A, the disulfide group constitutes a part of the main chain.

  An example in which AEDP is immobilized on isothiocyanate glass beads and the N-terminus of the peptide is modified using Compound A into which Br is introduced with 4-bromophenylalanine is shown, and this step is shown in the following formula. In the formula below, the peptide to be modified is pre-protected by guanidination. As shown in the following formula, the modified peptide is bound to the solid support at the N-terminus using 4-bromophenylalanine and AEDP as a linker.

  On the other hand, a group in which a group capable of specifically binding to the solid support is further bound to compound A may be used. As such a group, a functional group capable of binding to a portion other than the reaction point with the N-terminus of a protein or peptide in the main chain of the disulfide group-containing compound, and specifically binding to a fixed support. What has both the functional group which can be used can be used without specifically limiting. Examples thereof include a biotinyl group that can specifically bind to a support on which avidin is immobilized. Thus, what biotinyl group couple | bonded with the said compound A (biotinylated type compound A) is good to prepare by making the said compound A and a biotin acid derivative react. In biotinylated compound A, the disulfide group forms part of the main chain.

  An example of synthesizing biotinylated compound A is shown in the following formula. In the following formula, a compound having a disulfide bond (sulfosuccinimidyl-2- (biotinamido) ethyl-1,3′-dithiopropionate (sulfosuccinimidyl-2- (biotinamido) ethyl- 1,3′-dithiopropionate); available from Pierce as Sulfo-NHS-SS-Biotin), an amino acid having a Br atom (4-bromophenylalanine) is bound to biotinyl type compound A (10 -biotinamido-2- (4-bromobenzyl) -3-aza-4-oxo-7,8-dithiadecanoic acid).

  As the above formula shows, in the obtained compound A, the functional group capable of reacting with a protein or peptide is derived from the amino acid or amino acid derivative.

  An example of modifying the N-terminus of a peptide with biotinyl-based compound A is given, and this process is shown in the following formula. In the formula below, the peptide to be modified is pre-protected by guanidination.

  As described above, a protein or peptide in which the N-terminus is modified with compound A can be obtained. Since the method of the present invention does not introduce a sulfonic acid derivative directly into the N-terminus of a protein or peptide unlike the conventional method, it is more excellent in terms of reaction efficiency to the N-terminus.

[Fragmentation step and separation step]
In the method of the present invention, after the aforementioned modification step, prior to the cleavage step described below, fragmenting the protein or peptide (fragmentation step). In the fragmentation step, fragmentation breaks down into one N-terminal peptide fragment whose N-terminus is modified by Compound A and one or more other peptide fragments. The N-terminus of the N-terminal peptide fragment has a disulfide bond derived from compound A in the modification step described above.

  As a fragmentation method, chemical fragmentation or enzymatic digestion may be performed. When chemical fragmentation is performed, BrCN or the like may be used. When digesting with an enzyme, for example, since a guanidinized protein or peptide does not have a lysine residue, an endoprotease other than Lys-C can be used as the enzyme. In the method of the present invention, an enzyme that generates an N-terminal peptide fragment with a length that can be easily analyzed by a mass spectrometer or the like can be selected according to the protein to be analyzed. For example, trypsin, chymotrypsin, Glu-C and the like are used. This enzymatic digestion is performed using a conventional method.

  After performing the fragmentation step described above, the N-terminal peptide fragment and other peptide fragments are separated (separation step). In the separation step, the N-terminal peptide fragment may be selectively recovered. At this time, according to the nature of the modifying group of the N-terminal peptide fragment, those skilled in the art can appropriately determine the separation means.

  For example, the fragmentation step and the separation step when the immobilized compound A is used in the modification step are shown in the following formula.

  The above formula shows an example using an endoprotease that decomposes the C-terminus of the e residue and the C-terminus of the h residue in the fragmentation step. Once fragmented, N-terminal peptide fragments bound to the solid support and other peptide fragments not bound to the solid support are obtained. When the support is washed in the separation step, other peptide fragments are eluted, and the N-terminal peptide fragment can be recovered in a form bound to the support.

  Further, for example, the fragmentation step and the separation step in the case where the biotinylated compound A is used in the modification step are shown in the following formula.

  In the above formula, an example using an endoprotease that decomposes the C terminus of the e residue and the C terminus of the h residue is shown. Once fragmented, N-terminal peptide fragments with biotin and other peptide fragments without biotin are obtained. When this mixture is adsorbed on an avidin column packed with a solid support to which avidin is bound in the separation step, the N-terminal peptide fragment is adsorbed on the avidin column, and the other peptide fragments are not adsorbed on the column. Further, when the avidin column is washed with a phosphate buffer or the like, other peptide fragments are eluted from the column, and the N-terminal peptide fragment can be recovered in a form adsorbed on the avidin column.

  In this way, only the N-terminal peptide fragment can be recovered by the above process. The recovered N-terminal peptide fragment is bound with a modifying group derived from compound A used in the above-described modification step.

[Cleavage process]
Obtained by the upper Symbol separation step, the protein or peptide modified group is bonded N-terminally derived from the compound A is subjected to cleavage step described below. In this step, disulfide groups are converted into sulfonic acid groups by cleaving disulfide bonds.

  Disulfide bond cleavage can be oxidative or reductive. The oxidative cleavage can be performed by a usual method such as a formic acid oxidation method. Thereby, a sulfide group is converted into a sulfonic acid group. On the other hand, in reductive cleavage, a sulfide group is converted into a thiol group by reduction by a conventional method using dithiothreitol, and further, thiol group is converted into a sulfonic acid group by oxidation with performic acid or the like. Can be converted to

  For example, this step when the immobilized compound A is used in the modification step is shown in the following formula. As shown in the following formula, an N-terminal peptide fragment derivatized with a sulfonic acid is obtained by cleavage of a disulfide bond.

  Further, for example, this step when the biotinylated compound A is used in the modification step is shown in the following formula. As shown in the following formula, an N-terminal peptide fragment derivatized with a sulfonic acid is obtained by cleavage of a disulfide bond.

  As described above, a derivative having a sulfonic acid group introduced at the N-terminus is obtained. From the point of view of N-terminal peptide fragment recovery, the method of the present invention recovers the N-terminal peptide fragment by cleaving the disulfide bond, so that the N-terminal peptide fragment is recovered rather than by, for example, dissociating the avidin-biotin bond. The recovery rate of peptide fragments is high. In addition, the sulfonic acid derivative of the N-terminal peptide fragment obtained by the method of the present invention can be used for analysis by a mass spectrometer. At this time, the amino acid sequence can be determined by detecting the y ion series in the PSD analysis due to the presence of the N-terminal sulfonic acid group.

  Second, the present invention is a method for amino acid sequencing of proteins or peptides. This method is carried out by mass-analyzing a sulfonic acid derivative of an N-terminal peptide obtained by using the above-described method for derivatizing a protein or peptide with a sulfonic acid as a sample for mass spectrometry. At this time, in the N-terminal peptide derivatized with sulfonic acid, the y ion series is detected in the PSD analysis due to the presence of the sulfonic acid group. Thus, amino acid sequencing is efficient and easy.

  Furthermore, the N-terminal peptide of the sulfonic acid derivative of the N-terminal peptide subjected to mass spectrometry is detected as a characteristic peak due to the presence of atoms having a characteristic isotopic composition ratio. For this reason, it is very useful in that the N-terminal peptide to be analyzed can be distinguished and specified based on the characteristic peak from impurities other than the N-terminal peptide that may be present in the sample.

  Third, the present invention is Compound A having an atom having a characteristic isotope composition, a disulfide group, and a functional group capable of reacting with the N-terminus of a protein or peptide. That is, the compound A of the present invention is a compound that can modify the N-terminus of a protein or peptide. A schematic structure of Compound A is shown below. Here, a and b represent an organic group, X represents an atom having a characteristic isotope composition in mass spectrometry, and Y represents a functional group capable of reacting with the N-terminus of a protein or peptide. The atoms having a characteristic isotopic composition are as described above. The disulfide group constitutes a part of the main chain of the compound of the present invention.

  Compound A of the present invention includes those further bound to a solid support (immobilized compound A) and those further bound to a group capable of specifically binding to the solid support (for example, biotinylation). Type compound A) and the like. An example of compound A of the present invention is shown in the following structural formula. In the formula, a circle represents a solid support.

  In particular, the compound A of the present invention is useful for obtaining a sulfonic acid derivative having a sulfonic acid group introduced at the N-terminus of a protein or peptide, or for analyzing a protein or peptide by mass spectrometry, It is preferably used in the above-described method for derivatizing a protein or peptide with sulfonic acid or the method for determining the amino acid sequence of a protein or peptide by mass spectrometry. Details regarding Compound A of the present invention and the effects brought about by Compound A are as set forth in the description of the invention regarding these methods.

  Fourthly, the present invention is a protein or peptide in which the N-terminal amino group is modified with a group having a characteristic isotope composition and a disulfide group. The characteristic isotope composition is as described above. The disulfide group forms part of the main chain of the compound of the present invention. This compound can be obtained by the method described in the modification step in the sulfonic acid derivatization method of the protein or peptide already described. Therefore, in the compound of the present invention obtained by the method described in the modification step, a group having a characteristic isotope composition and a disulfide group further bonded to a solid support or a biotinyl group And those that are bound to.

  An example of the compound of the present invention bound to a solid support is shown in the following structural formula. In the formula, a to l represent amino acid residues having no side chain amino group, Hor represents a homoarginine residue, and a circle represents a solid support.

  Moreover, what is couple | bonded with the biotinyl group as another example of the compound of this invention is shown to the following structural formula. In the formula, a to l represent amino acid residues having no side chain amino group, and Hor represents a homoarginine residue.

The compound of the present invention is preferably used as an intermediate for obtaining a sulfonic acid derivative having a sulfonic acid group introduced at the N-terminus of a protein or peptide, or as an intermediate for analyzing a protein or peptide by mass spectrometry. . That is, it is useful as an intermediate in the above-described methods for derivatizing a protein or peptide with a sulfonic acid derivative or for determining the amino acid sequence of a protein or peptide by mass spectrometry. Details regarding the intermediates of the present invention and the effects brought about by the intermediates of the present invention are as set forth in the description of the invention regarding these methods.

<Example 1>
[1. Sulfosuccinimidyl-2- (biotinamido) ethyl-1,3'-dithiopropionate (sulfosuccinimidyl-2- (biotinamido) ethyl-1,3'-dithiopropionate; Sulfo-NHS-SS-Biotin manufactured by Pierce) ) And 4-bromophenylalanine]
Sulfo-NHS-SS-Biotin (Pierce) 6.1 mg and 4-bromophenylalanine (Aldrich) 12 mg were dissolved in 0.2 ml of 0.2 M phosphate buffer (pH 7.6) for 1 hour at 37 ° C. Reacted. Product A was isolated by liquid chromatography and evaporated to dryness.

[2. Coupling of compound A and peptide N-terminal amino group]
Laminin pentapeptide (manufactured by Peptide Institute, Inc.) was used as a model peptide. Laminin Pentapeptide, those peptides having the sequence of the tumor suppressor sites laminin is amidated, having the amino acid sequence of Tyr-Ile-Gly-Ser- Arg-NH 2. In the sequence, the C-terminal carboxyl group is amidated, and the amidated C-terminus is indicated with —NH ₂ . 1 nmol of this peptide and the above 1. 1 μl of a solution obtained by dissolving the total amount of Compound A obtained in 1 above in 100 μl of DMSO and 500 nmol of EDC were mixed in 10 μl of 0.2 M phosphate buffer (pH 7.6), and the reaction was performed at room temperature for 30 minutes. .

[3. Affinity purification using avidin beads, oxidative cleavage of disulfide bond by performic acid oxidation reaction, and N-terminal sulfonic acid derivatization]
In a suspension obtained by suspending 5 μl of avidin carrier (Promega) in 20 μl of 0.1 M phosphate buffer (pH 7.2), the above 2. The total amount of the reaction solution obtained in (1) was added, and the mixture was stirred at room temperature for 15 minutes. Thereafter, the avidin carrier was washed with 0.1 M phosphate buffer (pH 7.2). Next, add 10 μl of formic acid prepared in advance (0.95 ml of formic acid and 0.05 ml of 30% hydrogen peroxide solution and left at room temperature for 2 hours) at 4 ° C. Reacted for 1 hour. The supernatant of the reaction solution was recovered, and distilled water was added and immediately lyophilized to completely remove performic acid.

[4. Measurement with MS]
The freeze-dried product was redissolved in 20 μl of 0.1% by volume trifluoroacetic acid, desalted using ZipTip (manufactured by Waters), and subjected to MS measurement with a mass spectrometer Axima CFR plus (manufactured by Shimadzu Corporation). The spectrum obtained at this time is shown in FIG. In FIG. 1, the peak indicated by the arrow is one in which two peaks having a difference of 2 Da and having the same intensity are detected as characteristic pair peaks, that is, a peak of the N-terminal peptide fragment. Furthermore, PSD analysis was further performed on this peak to determine the amino acid sequence. The results obtained at this time are shown in FIG.

It is a MALDI-TOF MS spectrum obtained in this example. It is a PSD spectrum obtained in this example.

  SEQ ID NO: 1 is obtained by amidating a peptide having the sequence of a tumor suppressor site of laminin.

Claims (13)

By reacting compound A containing an atom exhibiting a characteristic isotope composition in mass spectrometry and a disulfide group with the N terminus of the protein or peptide, the protein or peptide whose N terminus is modified with compound A is obtained. A modification step to obtain;
In the modified protein or peptide, by converting to sulfonic acid groups by cleaving the disulfide bonds of the disulfide groups, see contains a cleavage step of sulfonic acid derivatives of the modified protein or peptide,
After the modification step and before the cleavage step,
By fragmenting the protein or peptide modified by the compound A, one N-terminal peptide fragment derived from the N-terminus of the protein or peptide and modified by the compound A, and other than the N-terminal peptide fragment A fragmentation step to obtain one or more other peptide fragments of
Separating the N-terminal peptide fragment selectively by separating it from the other peptide fragments,
In the cleavage step, a protein or peptide that obtains a sulfonic acid derivatized N-terminal peptide fragment by cleaving the disulfide bond derived from the compound A in the N-terminal peptide fragment and converting it to a sulfonic acid group Method for derivatizing sulfonic acid.   The method of derivatizing a protein or peptide according to claim 1, wherein the compound A has a functional group capable of reacting with the N-terminus of the protein or peptide.   The functional group capable of reacting with the N-terminus of the protein or peptide is a carboxyl group, isothiocyanate group, succinimidyloxycarbonyl group, p-nitrophenoxycarbonyl group, pentafluorophenoxycarbonyl group, and tetrafluorosulfophenoxy. The method for derivatizing a protein or peptide according to claim 2 selected from the group consisting of carbonyl groups.   The method for derivatizing a protein or peptide according to any one of claims 1 to 3, wherein the atom having the isotopic composition is Br, Cl or B.   The method for derivatizing a protein or peptide according to any one of claims 1 to 4, wherein the conversion to a sulfonic acid group in the cleavage step is performed by oxidative cleavage of the disulfide bond.   The protein or peptide according to any one of claims 1 to 4, wherein the conversion to a sulfonic acid group in the cleavage step is carried out by reductively cleaving the disulfide bond, followed by an oxidation reaction. Method of derivatization.   The method for derivatizing a protein or peptide according to any one of claims 1 to 6, further comprising a protecting step of protecting a side chain amino group of the protein or peptide before the modifying step.   The method for derivatizing a protein or peptide according to claim 7 wherein the side chain amino group is protected by guanidinolation in the protection step. In the modification step, first, an immobilized compound A in which a solid support is further bound to the compound A is prepared, and then the immobilized compound A is reacted with the N-terminus of the protein or peptide. The method for derivatizing a protein or peptide according to any one of claims 1 to 8 , wherein the protein or peptide is modified at the N-terminus with the immobilized compound A. In the modification step, first, an immobilized compound A in which a solid support is further bound to the compound A is prepared, and then the immobilized compound A is reacted with the N-terminus of the protein or peptide. Obtaining a protein or peptide having the N-terminus modified by the immobilized compound A,
In the fragmentation step, an N-terminal peptide fragment modified with the immobilized compound A and the other peptide fragment are obtained,
In the separation step, the N-terminal peptide is selectively recovered by eluting the other peptide fragments,
9. The protein or peptide according to any one of claims 1 to 8, wherein, in the cleavage step, a disulfide bond of the N-terminal peptide fragment is cleaved to obtain a sulfonic acid derivatized N-terminal peptide fragment. Method of acid derivatization. In the modification step, first, a biotinylated compound A in which a biotinyl group is further bonded to the compound A is prepared, and then the biotinylated compound A is reacted with the N-terminus of a protein or peptide, The method for derivatizing a protein or peptide according to any one of claims 1 to 8 , wherein the protein or peptide is modified at the N-terminus with a biotinylated compound A. In the modification step, first, a biotinylated compound A in which a biotinyl group is further bonded to the compound A is prepared, and then the biotinylated compound A is reacted with the N-terminus of a protein or peptide, Obtaining a protein or peptide whose N-terminus is modified by biotinylated compound A,
In the fragmentation step, an N-terminal peptide fragment modified with the biotinylated compound A and the other peptide fragment are obtained,
In the separation step, the N-terminal peptide fragment is selectively adsorbed on the support by adsorbing the N-terminal peptide fragment to a solid support on which avidin is immobilized and eluting the other peptide fragments. To collect
The protein or peptide according to any one of claims 1 to 8, wherein, in the cleavage step, a sulfonic acid derivatized N-terminal peptide fragment is obtained by cleaving a disulfide bond of the adsorbed N-terminal peptide fragment. To derivatize sulfonic acid. The protein or peptide sulfonic acid derivative obtained by using the method according to any one of claims 1 to 12 , wherein the protein is detected by detecting a specific peak based on the isotopic composition by mass spectrometry. Alternatively, a method for analyzing the amino acid sequence of a peptide.

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