JP4576972B2 - Method for mass spectrometry of sulfonic acid derivatized N-terminal peptide fragments of proteins or peptides - 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 for converting a protein or peptide into a sulfonic acid derivative, 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. However, in this method, since the reagent is allowed to act using a trypsin peptide, the N-terminal fragment cannot be selectively recovered, and only the N-terminal fragment of the protein or peptide cannot be converted to a sulfonic acid derivative.
Proceeding of the National Academy of Science of the United States of America, Volume 96, June 1999 , P. 7131-7136

  The 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 easily and efficiently analyzing a protein or peptide by mass spectrometry. For this purpose, there is provided a method that allows simple and efficient analysis by selectively recovering an N-terminal fragment of a protein or peptide as a sulfonic acid derivative and detecting a y-ion series in a mass spectrum. .

  A third object of the present invention is to provide an intermediate for efficiently derivatizing 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.

The present invention includes the following inventions.
1. The following inventions (1) to ( 12 ) relate to a method for mass spectrometry of an N-terminal peptide fragment of a protein or peptide by derivatization with a sulfonic acid.

(1) below shows a modification step with compound A, the cleavage step, have rows and mass spectrometry step, after the modification step, prior to the cleavage step, the fragmentation step and the separation step the line cormorants form .
(1) a modification step of obtaining a protein or peptide in which the N-terminus is modified with the compound A by reacting the N-terminus of the protein or peptide with a compound A containing a disulfide group;
By fragmenting the protein or peptide modified with the compound A, one N-terminal peptide fragment derived from the N-terminus of the protein or peptide and modified with 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;
By conversion to the N-terminal peptide disulfide bonds was cleaved with sulfonic acid groups of the disulfide group derived from the compound A in the fragment, the cleavage step Ru give sulfonic acid derivatized N-terminal peptide fragment,
Wherein said a step of acid derivatized N-terminal peptide fragment mass spectrometry, the protein or peptide acid derivatized N-terminal peptide fragment of a mass spectrometer.

The following (2) and (3) show the structure of Compound A in (1).
(2) The method for mass spectrometry of 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 mass spectrometry of a sulfonic acid derivatized N-terminal peptide fragment of a protein or peptide according to (2) selected from the group consisting of a fluorosulfophenoxycarbonyl group.

The following (4) and (5) show the cleavage step in (1).
(4) The sulfonic acid derivatization of the protein or peptide according to any one of (1) to (3), wherein the conversion to the sulfonic acid group in the cleavage step is performed by oxidative cleavage of the disulfide bond . For mass spectrometry of the prepared N-terminal peptide fragment .
(5) The protein according to any one of (1) to (3) above, wherein the conversion to a sulfonic acid group in the cleavage step is performed by reductively cleaving the disulfide bond and then performing an oxidation reaction. A method for mass spectrometry of a sulfonic acid derivatized N-terminal peptide fragment of a peptide .

The following (6) and (7) show forms in which a protection step is performed before the modification step in (1).
(6) The sulfonic acid derivatization of the protein or peptide according to any one of (1) to (5), further including a protecting step for protecting a side chain amino group of the protein or peptide before the modifying step . For mass spectrometry of the prepared N-terminal peptide fragment .
(7) A method for mass spectrometry of the sulfonic acid derivatized N-terminal peptide fragment of the protein or peptide according to (6), wherein the side chain amino group is protected by guanidinolation in the protection step.

The following ( 8 ) and ( 9 ) show forms using the immobilized compound A.
( 8 ) 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. by the said N-terminal by the immobilized type compound a to obtain a modified protein or peptide, wherein (1) the N-terminus which are sulfonic acid derivatives of the protein or peptide according to any one of (1) to (7) A method for mass spectrometry of peptide fragments .
( 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. 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 fragment is selectively recovered by eluting the other peptide fragments,
In the cleavage step, the by cleaving the disulfide bonds of the N-terminal peptide fragment to obtain a sulfonic acid derivatized N-terminal peptide fragment, wherein (1) to a protein or peptide according to any one of (7) A method for mass spectrometry of a sulfonic acid derivatized N-terminal peptide fragment .

The following ( 10 ) and ( 11 ) show forms using the biotinylated compound A.
( 10 ) 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, according to any one of (1) to ( 7 ) above, wherein the N-terminal peptide fragment is a sulfonic acid derivatized protein or peptide Of mass spectrometry .
( 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 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
In the cleavage step, the protein according to any one of (1) to (7) above, wherein a sulfonic acid derivatized N-terminal peptide fragment is obtained by cleaving a disulfide bond of the adsorbed N-terminal peptide fragment. A method for mass spectrometry of a sulfonic acid derivatized N-terminal peptide fragment of a peptide .

In the invention of the following (12), that determine the amino acid sequence of the sulfonic acid derivatized N-terminal peptide fragment.
( 12 ) In the step of mass spectrometry, the amino acid sequence of the N-terminal peptide fragment is determined. The sulfonic acid derivatized N-terminal peptide fragment of the protein or peptide according to any one of (1) to (11) Method for mass spectrometry .

2 . It follows invention relates to modified proteins or peptides.
A protein or peptide in which the N- terminal amino group is modified with a disulfide group-containing group.
Before SL disulfide group-containing group, further solid phase support is attached, before Symbol of the protein or peptide.
Before SL disulfide group-containing group is further linked with biotinyl groups, before Symbol of 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. In addition, 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 can be provided.

  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. Further, it is possible to provide a method that can be easily and efficiently analyzed by selectively recovering an N-terminal fragment of a protein or peptide as a sulfonic acid derivative and detecting a y-ion series in a mass spectrum. .

  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.

The present invention is a method for derivatizing a sulfonic acid by forming a sulfonic acid group in an N-terminal peptide fragment of a protein or peptide and performing mass spectrometry . In the method of the present invention, the N-terminus of a protein or peptide is modified with a compound containing a disulfide group (compound A), the fragmentation step and the separation step, and the disulfide group is cleaved and converted to a sulfonic acid group. a cleavage step of, and a mass spectrometry step, optionally, including a protection step prior to the modification step.

[Protection process]
In the method of the invention, 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 model of a peptide containing only lysine as an amino acid residue having a side chain amino group is protected by guanidinolation is shown, and this process is shown in the following formula 1. In the formula, a to l represent amino acid residues having no side chain amino group.

As shown in the above formula 1, the guanidinolation reaction converts a side chain amino group (—NH ₂ ) into a 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 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 substituted with the compound A (disulfide). Group-containing compound).

  Compound A in the method of the present invention is a compound having a functional group capable of reacting with the N-terminus of a protein or peptide and a disulfide group. As the functional group capable of reacting with the N-terminus, 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. Examples of compound A include AEDP (3-([2-aminoethyl] dithio) propionate hydrochloride), cystine derivatives and the like.

  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, diisopropylethylamine, 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 system 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 performed in a buffer solution at 0 to 70 ° C. for 0.1 to 24 hours.

  In the method of the present invention, the 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 to prepare immobilized compound A and the N-terminus of the peptide is modified using this immobilized compound A is shown in the following formula 2. In the following formula 2, the peptide to be modified is protected in advance by guanidinolation. As shown in the following formula 2, the modified peptide is bonded to the solid support at the N-terminus using AEDP as a linker.

  On the other hand, a compound in which a group capable of specifically binding to a solid support is bound to compound A may be used. Examples of such a group include a group capable of binding to a portion other than the reactive site with the N-terminus of the protein or peptide in the main chain of the compound A, and a function capable of specifically binding to a fixed support. Those having both groups can be used without any particular limitation. Examples thereof include a biotinyl group that can specifically bind to a support on which avidin is immobilized. A biotinyl group further bonded to compound A in this way (biotinylated compound) is preferably prepared by reacting compound A with a biotin acid derivative. In biotinylated compound A, the disulfide group forms part of the main chain.

  An example of modifying the N-terminus of the peptide by using AEDP to which a biotinyl group is bonded as the biotinylated compound A is given, and this process is shown in the following formula 3. In the following formula 3, the peptide to be modified is protected in advance by guanidinolation.

  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 results in degradation into one N-terminal peptide fragment derived from the N-terminus of the peptide to be sulfonated 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 using AEDP immobilized on a solid support as compound A in the modification step are shown in the following formula 4.

  Formula 4 above 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 when using AEDP to which a biotin derivative is bound as the biotinylated compound A in the modification step are shown in the following formula 5.

  In the above formula 5, 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]
The protein or peptide obtained by the modification step or the separation step, to which the N-terminal modified group derived from compound A is bound, is subjected to the 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 using AEDP immobilized on a solid support as the immobilized compound A in the modification step is shown in the following formula 6. As shown in the following formula 6, an N-terminal peptide fragment derivatized with a sulfonic acid is obtained by cleavage of a disulfide bond.

  Further, for example, this step when using AEDP to which a biotinyl group is bonded as the biotinylated compound A in the modification step is shown in the following formula 7. As shown in the following formula 7, 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 of a protein or peptide is obtained. From the point of view of N-terminal peptide fragment recovery, the method of the present invention recovers the N-terminal fragment by cleaving the disulfide bond, so that the N-terminal fragment rather than recovering by dissociating the avidin-biotin bond, for example. The recovery rate is high. Also preferred are sulfonic acid derivatives of the resulting protein or peptide by the method of the present invention is subjected to analysis by mass spectrometry, such as determining the amino acid sequence Ru cormorants performed. At this time, fragment ions are easily generated due to the presence of a sulfonic acid group in the protein or peptide.

Sulfonic acid derivatives of the resulting N-terminal peptides are used as a sample for mass spectrometric analysis are mass analyzed. When determining the amino acid sequence, the N-terminal peptide derivatized with sulfonic acid detects the y ion series in the PSD analysis due to the presence of the sulfonic acid group. Thus, amino acid sequencing is efficient and easy.

In the present invention, N-terminal amino group is a disulfide group-containing modified protein or peptide Ru obtained by group. 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 method of the present invention described above. The disulfide group-containing group in this compound is a group derived from compound A used in the method described in the modification step. As described in the modification step, this compound A can be used which is immobilized on a solid support or bonded to a biotinyl group. Accordingly, in the compound of the present invention obtained by the method described in the modification step, examples of the disulfide bond-containing group include those bonded to a solid support and those bonded to a biotinyl group. Examples of the compounds of the present invention are shown in the following structural formulas (I) and (II). In the formula, a to l represent amino acid residues, and a circle represents a solid support.

  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. That is, as described in the above-described cleavage step in the method of the present invention, the disulfide bond can be easily and efficiently converted into a sulfonic acid derivative.

  EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. Hereinafter, unless otherwise specified, “%” represents wt%. 1 to 4, the horizontal axis represents mass / charge (Mass / Charge), and the vertical axis represents the relative intensity (% Int.) Of the ion peak.

<Example 1> (For reference)
[Preparation of Compound A-bound Support]
10 mg of isothiocyanate glass beads (manufactured by Sigma) was washed with DMF, suspended in 100 μl of DMF, and 4.6 μmol of AEDP (3-([2-aminoethyl] dithio) propionate hydrochloride) and 0.5 μl of N-methylmorpholine was added and reacted at room temperature for 30 minutes. Then, it wash | cleaned with DMF and distilled water, and the obtained support body was used for the following modification reaction.

[Modification of peptide, cleavage of disulfide bond, and measurement with mass spectrometer]
Laminin pentapeptide (manufactured by Peptide Institute, Inc.) was used as a peptide sample. Laminin pentapeptide is an amidated peptide having a sequence of a tumor suppressor site of laminin, and has an amino acid sequence of Tyr-Ile-Gly-Ser-Arg-NH ₂ (SEQ ID NO: 1). In the sequence, the C-terminal carboxyl group is amidated, and the amidated C-terminus is indicated with —NH ₂ .

  3 mg of the support prepared as described above was suspended in 30 μl of distilled water, 500 nmol EDC and 500 nmol triethylamine were added and allowed to stand at room temperature for 30 minutes. Were added and the mixture was allowed to stand at room temperature for 30 minutes and reacted for 30 minutes. After completion of the reaction, the unreacted reagent was removed by washing with distilled water. Separately, formic acid and 30% by weight hydrogen peroxide water were mixed at a ratio of 19: 1 (volume ratio) and reacted at room temperature for 2 hours, 20 μl of formic acid prepared and sealed, and reacted at 4 ° C for 1 hour I let you. After completion of the reaction, the supernatant of the reaction solution was collected, distilled water was added and lyophilized. The sample was redissolved with a 0.1% by weight aqueous trifluoroacetic acid solution, desalted with ZipTipC18 (Millipore), and then analyzed by MALDI-TOF MS.

  FIG. 1 shows the spectrum of MALDI-TOF MS. As shown in FIG. 1, a sulfonic acid derivative of laminin pentapeptide was detected at 731.03 (m / z), and it was confirmed that the target product was produced. FIG. 2 shows a PSD spectrum in which 731.35 (m / z) is a precursor ion. As shown in FIG. 2, y-series ions (y2 to y5) including the C-terminus of the peptide were detected, and the structure from the N-terminus to 4 residues of the peptide was confirmed.

<Example 2>
Using Lysozyme as a protein sample, reductive alkylation and guanidinolation, N-terminal modification with disulfide group-containing compounds, trypsin digestion, adsorption of N-terminal fragments using avidin beads, and oxidative cleavage of disulfide bonds by formic acid oxidation By doing so, an example of selectively recovering the N-terminal fragment of Lysozyme as a sulfonic acid derivative is shown.

  Lysozyme (EC 3.2.1.17 from Chicken Egg White: manufactured by SIGMA) was dissolved in distilled water to a concentration of 100 pmol / μL. Of this, 10 μL was dropped onto a PVDF membrane (Polyvinylidene difloride menbrane: manufactured by Nippon Genetics) cut into a circle having a diameter of 8 mm and dried.

[Reduction-alkylation]
A PVDF membrane with a protein sample applied is placed in a 1.5 mL plastic tube, and a 100 mM NH ₄ HCO ₃ aqueous solution containing 10 mM dithiothreitol (DTT) is mixed with acetonitrile (8: 2 (v / v)). 1 ml was added and reacted at 56 ° C. for 1 hour. After completion of the reaction, the added mixture is removed from the tube, and 1 ml of a (8: 2 (v / v)) mixture of 100 mM NH ₄ HCO ₃ aqueous solution containing 55 mM iodoacetamide and acetonitrile is added to the tube. And allowed to react for 45 minutes at room temperature in the dark. After completion of the reaction, the PVDF membrane was taken out from the tube, placed in a beaker containing 100 ml of distilled water, and stirred for 10 minutes to wash the PVDF membrane. After washing, the PVDF membrane was dried.

[Guadininolation (protection of side chain amino group)]
The PVDF membrane obtained by the above reduction-alkylation treatment is put in a separately prepared 1.5 ml tube, and 7N ammonia water containing 0.85 M O-methylisourea hemisulfate (manufactured by Wako Chemical). And 1 ml of an (8: 2 (v / v)) mixture with acetonitrile was added and reacted at 65 ° C. for 30 minutes. The PVDF membrane was taken out from the tube, placed in a beaker containing 100 ml of distilled water, and stirred for 10 minutes to wash the PVDF membrane. After washing, the PVDF membrane was dried.

[Modification of protein N-terminus]
The PVDF membrane obtained by the above guanidinolation treatment was put into a separately prepared 1.5 mL tube, and 10 mM Sulfo-NHS-SS-biotin (sulfosuccinimidyl-2- (biotinamide) ethyl-1,3 -Dithiopropionate sulfosuccinimidyl-2- (biotinamido) ethyl-
1,3-dithiopropionate: 100 μL of a 0.2 M phosphate buffer solution (pH 7.2) and acetonitrile (8: 2 (v / v)) containing Pierce, containing the following structural formula (III): The reaction was carried out at 1 ° C. for 1 hour. After completion of the reaction, the PVDF membrane was taken out from the tube, placed in a beaker containing 100 mL of distilled water, and stirred for 10 minutes to wash the PVDF membrane. After washing, the PVDF membrane was dried.

[Trypsin digestion]
The PVDF membrane after the above modification treatment is placed in a separately prepared 1.5 mL digestion tube, and 100 mM NH ₄ HCO ₃ , 5 mM CaCl ₂ aqueous solution containing 1 μg trypsin and acetonitrile (2: 8 (v / v)) 100 μL of the mixture was added and digested overnight at 37 ° C.

[Extraction of digested peptide from PVDF membrane]
After the digestion, the reaction solution was taken out from the digestion tube and recovered in a separately prepared 1.5 ml recovery tube. Add 150 μL of a 0.1% TFA and acetonitrile (4: 6 v / v) mixture to the PVDF membrane remaining in the digestion tube and leave it at 60 ° C for 2 hours to extract the digested peptide from the PVDF membrane. went. The obtained extraction solution was recovered in the recovery tube. A series of operations of extracting the digested peptide and collecting the extracted solution was repeated again. The collected liquid was dried by a centrifugal concentrator. The obtained dry digested peptide was redissolved in 10 μL of 0.1 M phosphate buffer (pH 7.2).

For reference, 2 μL of this dissolved digested peptide solution was desalted using ZipTip ^™ C18 (MILLIPORE) and analyzed by MALDI MS. The mass spectrum obtained at this time is shown in FIG. As FIG. 3 shows, the peak (arrow) at 1037.66 (m / z) is a lysine residue guanidinylated and the N-terminus is a Bio-NH (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -CO group. (Bio: biotinyl group) modified N-terminal peptide fragment (Bio-NH (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -CO-Hor-Val-Phe-Gly-Arg
SEQ ID NO: 2; Bio: biotinyl group, Hor: homoarginine residue). Along with the N-terminal peptide fragment, a number of other trypsin-digested peptides were detected, namely trypsin-digested peptides derived from the internal sequence of reductive alkylated and guanidinoylated Lysozyme.

[Selective recovery of N-terminal peptide fragments]
(I) Preparation of avidin resin
Take 10 μL of SoftLink ^TM Softlease Avidin Resin (Promega) into a 1.5 ml tube, add 500 μL of 0.1 M phosphate buffer (pH 7.2), stir, precipitate the resin by centrifugation, and remove the supernatant . A series of operations of adding phosphate buffer, stirring, centrifuging, and removing the supernatant were repeated three times to wash and equilibrate the avidin resin.

(Ii) Selective recovery and elution of N-terminal fragment The remainder of the dissolved digested peptide was applied to the prepared avidin resin. The mixture was stirred at room temperature for 15 minutes to adsorb the N-terminal peptide fragment to the avidin resin. After the adsorption, the avidin resin was washed 3 times with 120 μL of 0.1 M phosphate buffer (pH 7.2) to remove peptides other than the N-terminal peptide fragment. Separately, formic acid and 30% by weight hydrogen peroxide were mixed at a ratio of 19: 1 (v / v) and left at room temperature for 2 hours to adjust formic acid. Thereafter, 10 μL of prepared formic acid was added to the avidin resin, and the mixture was stirred at 4 ° C. for 1 hour. The supernatant was recovered and lyophilized to obtain a sulfonic acid derivative of the dried N-terminal peptide fragment. It was. The dried sulfonic acid derivative was redissolved with 10 μL of 0.1% TFA aqueous solution, desalted using ZipTip ^™ C18, and analyzed by MALDI MS. The mass spectrum obtained at this time is shown in FIG. As FIG. 4 shows, a peak of 784.37 (m / z) corresponding to the sulfonic acid derivative of the N-terminal peptide fragment detected in FIG. 3 was specifically detected. From this, it was confirmed that the N-terminal peptide fragment of Lysozyme was selectively recovered as a sulfonic acid derivative.

3 is a MALDI-TOF MS spectrum obtained in Example 1. 3 is a PSD spectrum obtained in Example 1. 3 is a MALDI-TOF MS spectrum obtained for reference in Example 2. 3 is a MALDI-TOF MS spectrum obtained in Example 2.

SEQ ID NO: 1 is obtained by amidating a peptide having the sequence of a tumor suppressor site of laminin.
SEQ ID NO: 2 is part of the sequence of lysozyme that has undergone guanidinolation at the lysine residue and modification at the N-terminus.

Claims (12)

A modification step of obtaining a protein or peptide in which the N-terminus is modified with the compound A by reacting a compound A containing a disulfide group with the N-terminus of the protein or peptide;
By fragmenting the protein or peptide modified with the compound A, one N-terminal peptide fragment derived from the N-terminus of the protein or peptide and modified with 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;
By conversion to the N-terminal peptide disulfide bonds was cleaved with sulfonic acid groups of the disulfide group derived from the compound A in the fragment, the cleavage step Ru give sulfonic acid derivatized N-terminal peptide fragment,
Wherein said a step of acid derivatized N-terminal peptide fragment mass spectrometry, the protein or peptide acid derivatized N-terminal peptide fragment of a mass spectrometer. The method for mass spectrometry of a sulfonic acid derivatized N-terminal peptide fragment of 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. A method for mass spectrometry of a sulfonic acid derivatized N-terminal peptide fragment of a protein or peptide according to claim 2 selected from the group consisting of carbonyl groups. The sulfonic acid derivatized N-terminal of the protein or peptide according to any one of claims 1 to 3, wherein the conversion to a sulfonic acid group in the cleavage step is performed by oxidative cleavage of the disulfide bond. A method for mass spectrometry of peptide fragments . Conversion to sulfonic acid groups in the cleavage step, the disulfide bond was reductively cleaved, then, carried out by the oxidation reaction, sulfonic acid of a protein or peptide according to any one of claims 1 to 3 A method of mass spectrometric analysis of derivatized N-terminal peptide fragments . The sulfonated derivatized N-terminus of the protein or peptide according to any one of claims 1 to 5, further comprising a protection step of protecting a side chain amino group of the protein or peptide before the modification step. A method for mass spectrometry of peptide fragments . 7. The method for mass spectrometry of a sulfonic acid derivatized N-terminal peptide fragment of a protein or peptide according to claim 6, wherein in the protection step, the side chain amino group is protected by guanidinolation. 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. , wherein the N-terminus by the immobilized type compound a to obtain a modified protein or peptide, mass N-terminal peptide fragment which is sulphonic acid derivatives of the protein or peptide according to any one of claims 1-7 How to analyze . 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 fragment is selectively recovered by eluting the other peptide fragments,
The protein or peptide sulfone according to any one of claims 1 to 7, wherein in the cleavage step, a sulfonic acid derivatized N-terminal peptide fragment is obtained by cleaving a disulfide bond of the N-terminal peptide fragment. A method for mass spectrometry of acid-derivatized N-terminal peptide fragments . 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 N-terminal by biotinylated-type compound a to obtain a modified protein or peptide, the N-terminal peptide fragment which is sulphonic acid derivatives of the protein or peptide according to any one of claims 1 to 7 mass spectrometry Method. 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 7 , wherein a sulfonic acid derivatized N-terminal peptide fragment is obtained by cleaving a disulfide bond of the adsorbed N-terminal peptide fragment in the cleavage step. A mass spectrometric analysis of sulfonic acid derivatized N-terminal peptide fragments . The mass analysis of the sulfonic acid derivatized N-terminal peptide fragment of the protein or peptide according to any one of claims 1 to 11, wherein in the mass analysis step, an amino acid sequence of the N-terminal peptide fragment is determined. Method.

Images