JP4165501B2 - Method for measuring hydrophobic peptides by MALDI mass spectrometer - Google Patents

Description

  The present invention relates to the field of proteomic analysis using a mass spectrometer.

<Quantitative analysis>
In the field of proteome analysis (exhaustive protein analysis), PMF (peptide mass fingerprinting) analysis methods combining two-dimensional electrophoresis and mass spectrometers have been the mainstream, but the next-generation proteome is an alternative. As an analysis method, a method using stable isotopes has been devised. For example, Curr. Opinion Chem. Biol., 2003, 7, 70-77 describes quantitative proteomics using mass spectrometry using an ICAT (Isotope-Coded Affinity Tag) reagent. Furthermore, Rapid Commun. Mass Spectrom., 2003, 17, 1642-1650 and International Publication No. 2004/002950 pamphlet, the stable isotope of 2-nitrobenzenesulfenyl chloride (NBSCl) developed by the present inventors. Using labeled and unlabeled substances as labeling reagents, tryptophan residues of proteins or peptides are modified, and these peptides are obtained by enzymatic digestion with trypsin, and then using a hydrophobic chromatography column, nitrobenzenesulfenyl ( A method (hereinafter referred to as NBS method) is described in which peptides containing NBS) -labeled tryptophan are concentrated and quantitative analysis is performed.

<Sequence identification by MS / MS analysis>
In the PMF method, mass values of a plurality of peptide fragments obtained by enzymatic digestion of one protein are detected by a mass spectrometer, and these are compared with the mass values of theoretical fragments on a database to identify the protein. . On the other hand, in the NBS method, the labeled and concentrated peptides are subjected to MS / MS analysis to determine the sequence, and the protein containing such a sequence is searched on the database to identify the original protein. Therefore, in the NBS method, simultaneously with quantitative analysis by normal mass spectrometry, sequencing by MS / MS analysis is an essential step for protein identification.

The MS / MS analysis goes through three steps: 1) selection of a peptide called a precursor ion to be sequenced, 2) fragmentation of the selected precursor ion, and 3) detection of the mass value of the resulting fragment ion. Done. Mass spectrometers that can perform such MS / MS analysis include tandem-type two ion separation units for precursor ion selection and fragment ion detection, and decomposition by internal energy of ions ( Uses PSD (post-source decay), ion trap type that can perform MS analysis (MS ⁿ analysis) multiple times by supplementing ions, and ion cyclotron resonance (ICR) technology The FTICR type which uses the Fourier transform (FT; Fourier Transform) of the data obtained by using it as a mass spectrum signal can be mentioned.

  In a MALDI (Matrix Assisted Laser Desorption / Ionization) type mass spectrometer, a molecule called a matrix (generally an organic compound) mixed in the sample absorbs the energy of the laser beam and heats it. Is vaporized and ionized. The matrix surrounding the sample molecules is instantly vaporized, resulting in the sample molecules being released into the gas phase almost simultaneously. At this time, electrons and protons are transferred between the sample molecules and the matrix, and ionization of the sample molecules is achieved. At the same time, part of the energy is transferred to the sample molecules as internal energy.

  In this way, the matrix not only assists vaporization and ionization of the sample molecules, but also acts as a dispersant for the sample molecules, so the affinity between the matrix and the sample molecules is important for efficient ionization of the sample molecules. It is thought that there is. Therefore, an organic compound that is optimal for each sample to be measured has been searched and used. For example, α-cyano-4-hydroxycinnamic acid (4-CHCA) is widely used for measuring peptides.

  On the other hand, MALDI-IT (Matrix Assisted Laser Desorption / Ionization-Ion Trap) type, MALDI-IT-TOF (Matrix Assisted Laser Desorption / Ionization-Ion Trap-Time of Flight) type (for example, AXIMA-QIT (manufactured by Shimadzu Corporation)) In the MALDI-FTICR (Matrix Assisted Laser Desorption / Ionization-Fourier Transform Ion Cyclotron Resonance) type mass spectrometer, the sample molecules ionized by the MALDI method are once trapped in the cell, and then ion detection and selection, or MS / MS Analyze. For this reason, the time until the ions once generated reach the detector and are measured is longer by 2 to 3 orders of magnitude than that of a normal MALDI-TOF (Matrix Assisted Laser Desorption / Ionization-Time of Flight) type device. . Since ionized sample molecules have excessive internal energy, it is known that self-collapse occurs gradually. Actually, in the PSD measurement of the MALDI-TOF mass spectrometer, pseudo-MS / MS analysis is performed using this principle.

  Therefore, in MALDI-IT, MALDI-IT-TOF, and MALDI-FTICR type mass spectrometers, if large internal energy is received during ionization, many unwanted fragment ions are detected during measurement. is there. From this point, the use of 4-CHCA as a matrix gives excessive internal energy to the sample during ionization, so MALDI-IT, MALDI-IT-TOF and MALDI-FTICR mass spectrometry It is not preferable in the apparatus. For this reason, the matrix that is actually set as the default in the AXIMA-QIT apparatus is 2,5-dihydroxybenzoic acid (DHB), which is considered to have relatively little energy given to the sample molecules during ionization.

By Salvatore Sechi and Yoshiya Oda, Quantitative proteomics using mass spectrometry, “Current Opinion in Chemical Biology Biology) ", (UK), 2003, Volume 7, p. 70-77 Labeling of tryptophan residues by Hiroki Kuyama, Makoto Watanabe, Chikako Toda, Eiji Ando, Koichi Tanaka, and Osamu Nishimura An Approach to Quantitative Proteome Analysis by Labeling Tryptophan Residues, “Rapid Communications in Mass Spectrometry”, 2003, Vol. 17, p. 1642-1650 International Publication No. 2004/002950 Pamphlet

  Thus, in MALDI-IT type, MALDI-IT-TOF type, and MALDI-FTICR type mass spectrometers, DHB is mainly used as a matrix. However, when measuring a sample containing a hydrophobic peptide, the hydrophobic peptide cannot be efficiently ionized even if DHB is used.

  Therefore, an object of the present invention is to provide a method capable of efficiently ionizing hydrophobic peptides in a MALDI mass spectrometer, particularly a MALDI-IT, MALDI-IT-TOF, and MALDI-FTICR mass spectrometer. It is to provide.

  As a result of intensive studies, the present inventors have found that the object of the present invention can be achieved by using α-cyano-3-hydroxycinnamic acid or 3-hydroxy-4-nitrobenzoic acid as a matrix. The present invention has been completed.

The present invention includes the following inventions.
(1) A peptide is measured using α-cyano-3-hydroxycinnamic acid or 3-hydroxy-4-nitrobenzoic acid as a matrix by a mass spectrometer having a MALDI (matrix-assisted laser desorption / ionization) ion source. Method.
(2) When 3-hydroxy-4-nitrobenzoic acid is used as the matrix, a mixed matrix in which the 3-hydroxy-4-nitrobenzoic acid and α-cyano-4-hydroxycinnamic acid are combined is used. The method for measuring a peptide according to (1), wherein measurement is performed.

(3) The method for measuring a peptide according to (1) or (2), wherein the peptide is a peptide chemically modified with a hydrophobic compound.
(4) The method for measuring a peptide according to any one of (1) to (3), wherein the peptide is a peptide containing an amino acid residue modified with a sulfenyl compound.
(5) The method for measuring a peptide according to any one of (1) to (4), wherein the peptide is a peptide derivatized with 2-nitrobenzenesulfenyl chloride.

  Here, in this specification, a peptide is used with the meaning containing an oligopeptide, polypeptide, and protein.

(6) The method for measuring a peptide according to any one of (1) to (5), using a MALDI-IT (Matrix Assisted Laser Desorption / Ionization-Ion Trap) mass spectrometer.
(7) The method for measuring a peptide according to any one of (1) to (5), using a MALDI-IT-TOF (Matrix Assisted Laser Desorption / Ionization-Ion Trap-Time of Flight) mass spectrometer.
(8) The method for measuring a peptide according to any one of (1) to (5), using a MALDI-FTICR (matrix-assisted laser desorption / ionization-Fourier transform ion cyclotron resonance) mass spectrometer.

(9) The method for measuring a peptide according to any one of (1) to (8), wherein the matrix is used as a solution having a concentration of 1 mg / ml to a saturated concentration.
(10) The method for measuring a peptide according to any one of (2) to (9), wherein the α-cyano-4-hydroxycinnamic acid is used as a solution having a concentration of 1 mg / ml to a saturated concentration.
(11) The 3-hydroxy-4-nitrobenzoic acid solution and the α-cyano-4-hydroxycinnamic acid solution are used in combination at a volume ratio of 1:10 to 10: 1. ).

  According to the present invention, there is provided a method capable of efficiently ionizing a hydrophobic peptide in a MALDI mass spectrometer, particularly a MALDI-IT, MALDI-IT-TOF, or MALDI-FTICR mass spectrometer. be able to.

  The method of the present invention is a method for measuring a hydrophobic peptide sample using a mass spectrometer having a MALDI (Matrix Assisted Laser Desorption / Ionization) ion source. Until now, the ionization mechanism of the MALDI method has not been completely elucidated. The statements herein are based on the currently most accepted interpretation.

  In the present invention, the hydrophobic peptide / hydrophobic protein means that the amino acid residues of the peptide / protein are relatively high in the content of hydrophobic amino acids, particularly those with high hydrophobicity, those having the following groups: And the thing which received the following chemical modification. Examples of highly hydrophobic amino acids include tryptophan, isoleucine, tyrosine, phenylalanine, leucine, valine, and methionine. In addition, alanine, glycine, proline and the like may be regarded as hydrophobic amino acids.

In particular, the present invention is useful when measuring a hydrophobic peptide having a nitrobenzenesulfenyl group (NBS group: NO ₂ PhS group (Ph: phenylene group)) or a nitrophenyl group. Examples of such hydrophobic peptides include peptides containing a nitrotyrosine residue, a nitrophenylalanine residue, a tryptophan residue having an NBS group as a substituent, a cysteine residue having an NBS group as a substituent.

  The hydrophobic peptide may be obtained by chemically modifying the corresponding peptide with a hydrophobic compound. Here, the corresponding peptide includes a case where the peptide itself is hydrophobic. That is, the case where a hydrophobic peptide is derivatized into a peptide having higher hydrophobicity by chemical modification using a hydrophobic compound is also included. The hydrophobic compound is preferably a sulfenyl compound. A sulfenyl compound is a compound that specifically modifies tryptophan residues and cysteine residues in peptides or proteins. A particularly preferred example of the sulfenyl compound is 2-nitrobenzenesulfenyl chloride (NBS reagent). That is, as an example of a preferred hydrophobic peptide in the present invention, a peptide obtained by chemically modifying a peptide having a tryptophan residue or a cysteine residue using an NBS reagent can be mentioned.

  The method of the present invention is particularly useful as a mass spectrometer combined with a MALDI ion source in which the time until the generated ions reach the detector and are measured is longer than that of a normal MALDI-TOF type. Can be used. Such mass spectrometers include MALDI-IT (Matrix Assisted Laser Desorption / Ionization-Ion Trap) Mass Spectrometer, MALDI-IT-TOF (Matrix Assisted Laser Desorption / Ionization-Ion Trap-Time of Flight) Mass Spectrometry. Examples include an apparatus MALDI-FTICR (Matrix Assisted Laser Desorption / Ionization-Fourier Transform Ion Cyclotron Resonance) type mass spectrometer.

  In the present invention, α-cyano-3-hydroxycinnamic acid (3-CHCA; the following formula I) or 3-hydroxy-4-nitrobenzoic acid (3H4NBA; the following formula II) is used as the matrix.

  These compounds can be appropriately used by those skilled in the art for the purpose of being used as a matrix for mass spectrometry. For example, these compounds are preferably used as a solution. Although it does not specifically limit as a density | concentration of a solution, For example, it can use as a solution of 1 mg / ml-saturation concentration.

  As a solvent used for preparing such a solution, it is preferable to use an acetonitrile aqueous solution, a trifluoroacetic acid (TFA) aqueous solution, or an acetonitrile-trifluoroacetic acid (TFA) aqueous solution. When an acetonitrile aqueous solution or an acetonitrile-TFA aqueous solution is used, the concentration of acetonitrile is not particularly limited, but 90% or less, preferably about 50% can be used. When a TFA aqueous solution or acetonitrile-TFA aqueous solution is used, the concentration of TFA is not particularly limited, but 1% or less, preferably about 0.1% can be used.

  When 3-CHCA is used as a matrix, it is not particularly limited by dissolving it in the above-mentioned solvent. For example, it can be used as a matrix solution having a concentration of 1 mg / ml to a saturated concentration, preferably 10 mg / ml. Further, when 3H4NBA is used as a matrix, it is not particularly limited by dissolving it in such a solvent. For example, it can be used as a matrix solution of 1 mg / ml to a saturated concentration, preferably as a saturated matrix solution.

  In the present specification, the standard of the amount expressed in% is v / v% unless otherwise specified.

By using the above matrix, there are the following advantages.
First, as mentioned above, the MALDI-IT mass spectrometer, the MALDI-IT-TOF mass spectrometer, and the MALDI-FTICR mass spectrometer are used until the generated ions reach the detector and are measured. Is longer than normal MALDI-TOF type. In addition, the matrix is heated and vaporized / ionized by the energy of the received laser, but a part of the energy becomes the internal energy of the sample molecule and gradually causes the self-decay of ions. That is, as the time until the generated ions reach the detector and are measured is longer, the self-decay progresses, and as a result, many unwanted fragment ions are detected. The matrix used in the present invention is considered to have a smaller internal energy given to the measurement molecule at the time of ionization than a matrix in a conventional method (for example, a method in which 4-CHCA is used alone as a matrix). Therefore, as described above, even when the generated ions reach the detector and are measured for a long time until they are used in the mass spectrometer, the progress of self-decompression of the generated ions can be suppressed.

  For this reason, the present invention is particularly useful for a mass spectrometer having a relatively long time from ionization to ion detection, such as an ion trap mass spectrometer. Therefore, it goes without saying that the present invention is also useful in a mass spectrometer having no ion trap.

  Furthermore, as mentioned above, the affinity between the matrix and the sample molecules is an important factor during ionization. Accordingly, it is considered that a highly hydrophobic matrix is suitable for a sample having a high degree of hydrophobicity, and a highly hydrophilic matrix is suitable for a sample having a high degree of hydrophilicity. Matrix 3-CHCA and 3H4NBA used in the present invention are considered to have a higher degree of hydrophobicity than conventional matrices (for example, DHB). On the other hand, since the peptide sample molecules measured in the present invention also have a high degree of hydrophobicity, it is considered that the affinity with 3-CHCA or 3H4NBA as a matrix is high. As a result, it is considered that the peptide sample and the matrix can easily form uniform microcrystals, and efficient ionization can be achieved.

  In the present invention, when 3-hydroxy-4-nitrobenzoic acid (3H4NBA) is used as the matrix, a mixed matrix in which 3H4NBA is combined with α-cyano-4-hydroxycinnamic acid (4-CHCA; Formula III below) It is preferable to use as. 4-CHCA is a compound that is widely used as a matrix in mass spectrometry of peptides. (Hereinafter, in this specification, a matrix that is used alone without using 4-CHCA in combination and a mixed matrix that is used in combination with 4-CHCA may be simply referred to as a matrix.)

The ratio of the combination of 3H4NBA and 4-CHCA is not particularly limited, but can be combined in the following quantitative relationship, for example.
For example, 3H4NBA can be prepared at concentrations as already described. That is, as a solution having a concentration of 1 mg / ml to a saturated concentration, for example, an acetonitrile aqueous solution, a TFA aqueous solution, or an acetonitrile-TFA aqueous solution is used as a solvent, it can be prepared as a 3H4NBA solution having a concentration of 1 mg / ml to a saturated concentration, preferably a saturated concentration.
On the other hand, 4-CHCA can be prepared at a concentration conventionally used. For example, it can be prepared as a solution of 1 mg / ml to a saturated concentration. When an acetonitrile aqueous solution, a TFA aqueous solution or an acetonitrile-TFA aqueous solution is used as a solvent, as in the case of using 3H4NBA, it can be prepared as a 4-CHCA solution having a concentration of 1 mg / ml to a saturated concentration, preferably 10 mg / ml.
Both solutions prepared as described above are preferably used in a volume ratio of 1:10 to 10: 1, more preferably 1: 3 to 3: 1, for example 1: 1.

  The conventional matrix 4-CHCA causes self-disintegration of the measurement sample in measurements with MALDI devices such as MALDI-IT, MALDI-IT-TOF, and MALDI-FTICR devices that require a long time from ionization to ion detection. Despite its drawbacks, it has excellent measurement sensitivity and has the advantage that it is easy to find the optimum spot to which a laser is applied in a mass spectrometry sample.

  On the other hand, the matrix 3H4NBA of the present invention has the advantages that, as already described, the progress of self-degradation of the measurement sample can be suppressed and efficient ionization of the hydrophobic sample can be achieved. Then, when the matrix 3H4NBA of the present invention is combined with 4-CHCA, a synergistic effect of the advantages of both matrices is achieved. In other words, while maintaining the advantages of suppressing the self-disintegration of the measurement sample possessed by 3H4NBA alone and achieving ionization of the hydrophobic sample, the high sensitivity detection capability of 4-CHCA further accompanies, and mass spectrometry with higher analytical efficiency can be performed. It becomes possible to do.

  According to the present invention, the range of methods capable of performing sequence identification of hydrophobic peptides (for example, NBS-labeled peptides) has been greatly expanded. In the prior art, MS / MS analysis using a mass spectrometer using ESI (electrospray ionization) and PSD analysis using a MALDI mass spectrometer were used. However, according to the present invention, CID (collision-induced dissociation) is performed. MS / MS analysis using a MALDI-IT mass spectrometer, a MALDI-IT-TOF mass spectrometer, or a MALDI-FTICR mass spectrometer, which can be started, is now possible. Furthermore, since CID can cause fragmentation more efficiently than PSD, it has become possible to improve the peptide identification rate.

  EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. As already stated, the standard for the quantity expressed in% is v / v% unless otherwise specified. In the examples, labeling with the NBS reagent may be referred to as modification or NBS modification.

[Example 1]
In this example, an NBS-labeled peptide (a mixture of a peptide modified with an NBS (heavy) reagent labeled with six stable isotopes ¹³ C and a peptide modified with an unlabeled NBS (light) reagent) Is used as a measurement sample, and the matrix 3-CHCA (α-cyano-3-hydroxycinnamic acid, formula I) and 3H4NBA (3-hydroxy-4-nitrobenzoic acid, formula II) of the present invention are compared with conventional ones for comparison. Was measured with a mass spectrometer using the matrix DHB (2,5-dihydroxybenzoic acid, formula IV).

The measurement sample was prepared as follows.
Two types of purified proteins (ovalbumin, glyceraldehyde-3-phosphate dehydrogenase, lysozyme, α-lactalbumin; all manufactured by SIGMA) were mixed at 25 μg each to give a total of 100 μg. According to the protocol of “ ¹³ CNBS Isotope Labeling Kit” (manufactured by Shimadzu Corporation), except that urea with a final concentration of 8M was used as a denaturing agent and each mixture was solubilized and the NBS-modified sample mixture was resolubilized. A measurement sample was prepared. That is, one of the two mixtures is NBS Reagent (heavy) (2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride) and the other is NBS Reagent (light) (2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride). ), And both modified samples were mixed, desalted, resolubilized with urea, reduced / alkylated, and trypsin digested.

  The sample was then lyophilized, suspended in 500 μl of 0.1% aqueous trifluoroacetic acid (TFA), and loaded onto a HiTrap Phenyl FF (high sub) column (Amersham Biosciences) equilibrated with 0.1% aqueous TFA. . The column was washed with 3 ml of 0.1% aqueous TFA and then eluted with 1 ml of 0.1% aqueous TFA containing 10% acetonitrile. Subsequently, the acetonitrile concentration of the TFA aqueous solution used for elution was changed to 15, 20, 25, 30, 35, and 40% in this order, and elution was performed in the same manner. After elution, the fraction eluted with 0.1% TFA aqueous solution containing 10% acetonitrile was lyophilized, resuspended in 50 μl 0.1% TFA aqueous solution, and desalted with ZipTip (μC18). In this way, a measurement sample was obtained.

  Examples of the matrix include 3-CHCA (α-cyano-3-hydroxycinnamic acid, formula I) and 3H4NBA (3-hydroxy-4-nitrobenzoic acid, formula II) of the present invention, and a conventional matrix for comparison. Three types of DHB (2,5-dihydroxybenzoic acid, formula IV) were prepared.

  A matrix prepared as follows was used. 3-CHCA, 3H4NBA and DHB were dissolved in a 50% acetonitrile aqueous solution containing 0.1% TFA, respectively. DHB and 3-CHCA were each 10 mg / ml solution, and 3H4NBA was a saturated solution.

  0.5 μl each of the measurement sample solution and the matrix solution obtained as described above were mixed and dropped onto the mass plate. The solution was dried and then measured with an AXIMA-QIT apparatus (MALDI-IT-TOF type mass spectrometer, manufactured by Shimadzu Corporation).

The MS spectrum obtained from the AXIMA-QIT apparatus is shown in FIG. In FIG. 1, the horizontal axis represents mass / charge (Mass / Charge), and the vertical axis represents the relative intensity of ions. In addition, (a) is the spectrum of NBS modified peptide when DHB is used for the matrix, (b) is the spectrum of NBS modified peptide when 3-CHCA is used for the matrix, (c) is the matrix of 3H4NBA. It is a spectrum of an NBS modified peptide when used for. In the figure, (i), (ii) and (iii) show pair peaks of peptides modified with NBS. Each paired peak consists of two labeling reagents NBS Reagent (heavy) (2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride) and NBS Reagent (light) (2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride). Detected as a set of peaks having a difference of m / z = 6 corresponding to a mass difference of. The theoretical mass value, sequence, and derived protein corresponding to each pair peak are as follows.

(i) 1119.53, 1204.55 (m / z), GTDVQAWIR (SEQ ID NO: 1), lysozyme
(ii) 1244.51, 1250.53 (m / z), LDQWLCEK (SEQ ID NO: 2), α-lactalbumin
(iii) 2011.95, 2017.97 (m / z), ELINSWVESQTNGIIR (SEQ ID NO: 3), ovalbumin

  FIG. 2 is a fragment spectrum when MS / MS analysis is performed after selectively trapping ions (GTDVQAWIR) corresponding to 1118.53 m / z of the pair peak (i) in FIG. In FIG. 2, the horizontal axis represents mass / charge (Mass / Charge), and the vertical axis represents the relative intensity of ions.

[Example 2]
(A) Mass spectrometry measurement using a mass spectrometer having an ion trap and a mass spectrometer having no ion trap

  A mixture of a peptide modified with NBS reagent and an unmodified peptide was used as a measurement sample, and the mixed matrix of the present invention, ie, 3H4NBA (3-hydroxy-4-nitrobenzoic acid, formula II) and conventional matrix 4-CHCA ( Using a mixture with α-cyano-4-hydroxycinnamic acid, formula III), measurements were taken with a mass spectrometer.

The measurement sample was prepared as follows.
Two types of purified proteins (ovalbumin, glyceraldehyde-3-phosphate dehydrogenase, lysozyme, α-lactalbumin; all manufactured by SIGMA) were mixed at 25 μg each to give a total of 100 μg. The protocol of “ ¹³ CNBS Isotope Labeling Kit” (manufactured by Shimadzu Corporation) was followed except that each mixture was denatured using urea having a final concentration of 8M as a denaturant. That is, one is labeled with NBS Reagent (heavy) (2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride) and the other is labeled with NBS Reagent (light) (2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride). Unlabeled modification, mixing of both modified samples, desalting, resolubilization with urea, reduction, alkylation, and trypsin digestion were performed. The sample after digestion was desalted with ZipTipμ-C18 and eluted with 4 μl of 50% acetonitrile aqueous solution containing 0.1% TFA was used as a measurement sample. Of this, 0.5 μl was applied on a mass plate.

  A matrix prepared as follows was used. 3H4NBA and 4-CHCA were dissolved in 50% acetonitrile aqueous solution containing 0.1% TFA as a solvent. 3H4NBA was a saturated solution, and 4-CHCA was a 10 mg / ml solution. The solution thus prepared was mixed at a volume ratio of 1: 1 to obtain a matrix mixed solution. MALDI-IT-TOF mass spectrometer with ion trap (AXIMA-QIT, Shimadzu Corporation) after adding 0.5 μl of the matrix mixed solution to the mass plate with the measurement sample applied in advance and drying And a MALDI-TOF mass spectrometer (AXIMA-CFR plus, manufactured by Shimadzu Corporation) without an ion trap.

The MS spectra obtained at this time are shown in FIGS. 3 (a-1) and 3 (a-2). In these figures, the horizontal axis represents mass / charge (m / z), and the vertical axis represents the relative intensity of ions (% Int.). In addition, (a-1) is the spectrum obtained by measuring with AXIMA-QIT with an ion trap, and (a-2) is obtained by measuring with AXIMA-CFR plus without an ion trap. It is a spectrum. Further, in these figures, the peak pair marked with an arrow indicates that the peptide is an NBS-modified peptide. Each paired peak is composed of two modifying reagents NBS Reagent (heavy) (2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride) and NBS Reagent (light) (2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride). It has a difference of m / z = 6 corresponding to the mass difference.

  As can be seen by comparing the spectra of FIG. 3 (a-1) and FIG. 3 (a-2) with each other, almost the same spectra were obtained in both cases. The fact that almost the same spectrum can be obtained by a mass spectrometer with an ion trap and a mass spectrometer without an ion trap means that the self-disintegration of the measurement sample is suppressed in the measurement of the mass spectrometer with an ion trap. Indicates. That is, when the matrix mixture of the present invention is used, in the conventional measurement with an ion trap type mass spectrometer (ie, a mass spectrometer having a relatively long time from ionization to ion detection) using 4-CHCA alone as a matrix. It was found that the self-disintegration of the measurement sample that had occurred was suppressed.

  (B) Mass spectrometric measurement using a matrix of 3H4NBA alone and a mixed matrix of 3H4NBA and 4-CHCA

  Using the same measurement sample as in the above (a), measurement was performed with a mass spectrometer using a matrix of the present invention, which is a matrix of 3H4NBA alone, and a mixed matrix of 3H4NBA and 4-CHCA.

  The same measurement sample as in the above (a) was diluted 1000 times with a 0.1% TFA aqueous solution, 0.5 μl of which was applied onto a mass plate.

As the matrix, a matrix using 3H4NBA alone and a mixed matrix of 3H4NBA and 4-CHCA were used.
For the matrix using 3H4NBA alone, 3H4NBA was dissolved in 50% acetonitrile aqueous solution containing 0.1% TFA to obtain a saturated solution.
A mixed matrix of 3H4NBA and 4-CHCA was prepared by the same method as in (a) above.

  A 3H4NBA solution and a mixed solution of 3H4NBA and 4-CHCA were added to the mass plate on which the measurement sample had been applied in advance, and after drying, a MALDI-TOF mass spectrometer (AXIMA-CFR plus, manufactured by Shimadzu Corporation).

The MS spectra obtained at this time are shown in FIGS. 3 (b-1) and 3 (b-2). In these figures, the horizontal axis represents mass / charge (m / z), and the vertical axis represents the relative intensity (%) of ions. In addition, (b-1) is a spectrum obtained by using 3H4NBA and 4-CHCA as a matrix and (b-2) is a spectrum obtained by using 3H4NBA as a matrix. Furthermore, the peak pairs marked with arrows in these figures indicate that they are of NBS-modified peptides. Each paired peak consists of two labeling reagents NBS Reagent (heavy) (2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride) and NBS Reagent (light) (2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride). M / z = 6, corresponding to a mass difference of

  As can be seen by comparing the spectra of (b-1) and (b-2) in FIG. 3, the paired peaks of the NBS-modified peptide were detected with higher sensitivity in (b-1) than in (b-2). ing. This indicates that detection can be performed with higher sensitivity by further mixing 4-CHCA with 3H4NBA as a matrix. In other words, the characteristics of 3H4NBA that “can detect a hydrophobic sample in mass spectrometry” remain unchanged, and 4-CHCA can easily find the optimum spot to which a laser is applied. It was confirmed that the feature that “good measurement can be performed” was added. That is, the feature of 3H4NBA is that it can suppress self-disintegration of the sample even if it is measured with an ion trap MALDI mass spectrometer with a relatively long time from ionization to ion detection, and 4-CHCA It was confirmed that “the ability to make a highly sensitive measurement” was achieved at the same time in the state of “Easy to find the optimum spot to shine laser”.

In Example 1, it is the comparative MS spectrum (a) obtained by the conventional method, and the MS spectra (b) and (c) obtained by the method of the present invention. 2 is an MS / MS spectrum obtained in Example 1. In Example 2 (a), the MS spectrum (a-1) obtained by measuring with AXIMA-QIT having an ion trap, and the MS obtained by measuring with AXIMA-CFR plus without an ion trap In spectrum (a-2) and Example 2 (b), MS spectrum (b-1) obtained by mixing 3H4NBA and 4-CHCA as a matrix and 3H4NBA as a matrix This is an MS spectrum (b-2) obtained by.

Claims (11)

  A method of measuring a peptide using α-cyano-3-hydroxycinnamic acid or 3-hydroxy-4-nitrobenzoic acid as a matrix by a mass spectrometer having a MALDI (matrix-assisted laser desorption / ionization) ion source.   When 3-hydroxy-4-nitrobenzoic acid is used as the matrix, measurement is performed using a mixed matrix in which the 3-hydroxy-4-nitrobenzoic acid and α-cyano-4-hydroxycinnamic acid are combined. The method for measuring the peptide according to claim 1.   The method for measuring a peptide according to claim 1 or 2, wherein the peptide is a peptide chemically modified with a hydrophobic compound.   The method for measuring a peptide according to any one of claims 1 to 3, wherein the peptide is a peptide containing an amino acid residue modified with a sulfenyl compound.   The method for measuring a peptide according to any one of claims 1 to 4, wherein the peptide is a peptide derivatized with 2-nitrobenzenesulfenyl chloride.   The method for measuring a peptide according to any one of claims 1 to 5, using a MALDI-IT (matrix-assisted laser desorption / ionization-ion trap) mass spectrometer.   The method for measuring a peptide according to any one of claims 1 to 5, using a MALDI-IT-TOF (Matrix Assisted Laser Desorption / Ionization-Ion Trap-Time of Flight) mass spectrometer.   The method for measuring a peptide according to any one of claims 1 to 5, using a MALDI-FTICR (matrix-assisted laser desorption / ionization-Fourier transform ion cyclotron resonance) mass spectrometer.   The method for measuring a peptide according to any one of claims 1 to 8, wherein the matrix is used as a solution having a concentration of 1 mg / ml to a saturated concentration.   The method for measuring a peptide according to any one of claims 2 to 9, wherein the α-cyano-4-hydroxycinnamic acid is used as a solution having a concentration of 1 mg / ml to a saturated concentration. The solution of 3-hydroxy-4-nitrobenzoic acid and the solution of α-cyano-4-hydroxycinnamic acid are used in combination at a volume ratio of 1:10 to 10: 1. Method for measuring the peptide.