JP2010216945A - Analytical method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for analyzing high molecular substances such as polypeptides, proteins, and glucose without having to mix them with a matrix substance. <P>SOLUTION: An analyte of proteins and polypeptides or cells and biological tissue containing them is mounted to a resin sheet made of, for example, a cyclic polyolefin, an aromatic nylon, and a polycarbonate. The analyte mounted on the resin sheet is irradiated with an infrared laser beam to ionize the analyte and perform detection by mass analysis. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to analytical methods, and more specifically to laser desorption ionization mass spectrometry.

  In recent years, comprehensive analysis of proteomics that comprehensively analyzes the whole picture of a protein group (proteome) expressed from a specific gene, and analysis of metabolites including low molecular weight compounds such as organic acids and amino acids in the living body Metabolomics to be analyzed is attracting attention.

  The application of mass spectrometry is expected for such proteomics and metabolomics, as well as in vivo imaging for image processing of proteins in living bodies, and discovery of biomarkers. Matrix-assisted laser desorption / ionization (MALDI) is regarded as the most promising means for analyzing in vivo proteins and polypeptides by mass spectrometry (for example, Non-Patent Document 1). .

In this MALDI method, (1) a compound (matrix solution) that absorbs laser light and a trace amount of sample solution are mixed and dried on a sample stage; (2) a pulse laser is applied to the dried sample. And the step of ionizing the sample to perform mass analysis. As the pulse laser, a CO ₂ laser or Er: YAG laser that emits laser light in the infrared region is preferably used rather than a laser light source that emits laser light in the ultraviolet region such as a nitrogen laser or an Nd: YAG laser. This is because molecules constituting proteins have a very high vibration absorption coefficient in the infrared wavelength region, and it is considered that mass spectrometry of biological tissues can be selectively realized by using this. (Non-Patent Document 1).

Sachiko Suzuki et al., O plus E Vol.30, No.4 p348〜353,2008

  Until now, as compounds (matrix substances) used in the above matrix solution, for example, glycerin, sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihyroxybenzoic acid (DHB), etc. Molecular compounds have been used.

  However, in the above method, the low molecular compound used as the matrix material is also ionized at the same time, and in addition to the molecular ions of the sample and the fragment ions thereof, the molecular ions of the low molecular compounds and the fragment ions of the low molecular compounds are detected. For this reason, there has been a problem that the sensitivity of molecular ions and fragment ions of the target sample is lowered and the resolution is lowered.

  In addition, in biological samples containing proteins such as cells and biological tissues, fragment ions and amino acid ions in the cells and tissues are detected, so that analysis cannot be performed sufficiently. It cannot be said that it is a sufficient method for analysis.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide means for analyzing a high molecular weight substance such as a polypeptide, protein, or sugar by mass spectrometry without mixing with a matrix substance. There is.

  The analysis method of the present invention is a method for analyzing an analyte by laser desorption ionization mass spectrometry, wherein (1) the step of placing the analyte on a resin sheet; and (2) the resin sheet And analyzing the analyte by irradiating the placed analyte with an infrared laser, ionizing the analyte and detecting it by mass spectrometry.

  The analysis method of the present invention can detect molecular ions of proteins and peptides to be measured simply by placing an analyte on a resin sheet.

It is a schematic perspective view which shows an example of the resin sheet used in the analysis method of this invention. (A)-(h) is a figure which shows the absorption spectrum of the resin sheet which can be used in the analysis method of this invention, respectively. (A)-(h) is the chemical structural formula of resin used for the resin sheet of FIG. 1, respectively. (A)-(h) is a mass spectrum which shows the result of the mass spectrometry of angiotensin II obtained using the resin sheet shown in FIG. 1, respectively. It is a mass spectrum which shows the result of the mass spectrometry of angiotensin II obtained using the resin sheet of polyacrylic acid. It is a mass spectrum which shows the result of the mass spectrometry of Tuftsin, Comprising: (a) is a mass spectrum analyzed immediately after application | coating of a sample solution, (b) is a mass spectrum analyzed 24 hours after application | coating. It is a mass spectrum which shows the result of the mass spectrometry of Tuftsin when a resin sheet is used. It is a mass spectrum which shows the result of the mass spectrometry of Tuftsin when not using a resin sheet.

  The analysis method of the present invention is a method for analyzing an analyte by laser desorption ionization mass spectrometry, wherein (1) the step of placing the analyte on a resin sheet; and (2) the resin sheet Irradiating the placed analyte with an infrared light laser, ionizing the analyte and detecting it by mass spectrometry, and analyzing the analyte.

  The analysis method of the present invention includes (1) a step of placing an analyte on a resin sheet when performing mass spectrometry.

  As in the conventional MALDI method, the analyte to be analyzed in the present invention includes proteins, polypeptides, peptides, and sugars that have been purified in advance. Samples containing so-called contaminants other than the target analyte, such as peptides and sugars, such as crude extracts from cells and biological tissues, cells and biological tissues themselves, and the term analyte are analyzed. It is used in a broader sense than the analyte (target substance) that is the object.

  In the present invention, it is not necessary to strictly distinguish proteins, polypeptides, and peptides, and any compound (peptide) in which two or more amino acids are bound by peptide bonds is the subject of analysis. Among these, peptides having a molecular weight (M.W.) of 100 or more, more preferably high molecular substances such as peptides (proteins) or sugars of 500 or more are analyzed well.

The analysis method of the present invention is characterized in that a resin sheet is used as an alternative to the matrix material in the conventional MADLI method. That is, in the analysis method of the present invention, the resin sheet supports mass analysis of the analyte as a matrix substance. Therefore, the resin sheet is not particularly limited as long as the sheet can support this. In the MADLI method, a matrix material that absorbs irradiated laser light, weakens excess energy of the laser light, and weakens cohesion between sample molecules is used. In the present invention, it is considered that the resin sheet performs the same function as this matrix agent. Therefore, in the present invention, a resin sheet in which atoms or molecular groups that absorb photons corresponding to the wavelength of the laser beam irradiated into the sample molecules and excite vibrational motion is preferable is considered preferable. When protein, peptide, or sugar is the target of analysis, OH stretching mode or C-H stretching mode or C-O stretching mode or C = O stretching mode or wavelength around 6 μm. Is considered as a target (see Non-Patent Document 1). If it does so, it is thought that the resin sheet which has an absorption peak in the vicinity of such a wavelength fulfill | performs the function as a matrix agent. For example, a polyacrylic acid resin sheet will be described. When a side chain carboxyl group (—CO (OH) group) is esterified with a methyl group and the side chain is —CO (OCH ₃ ), the sample molecule is Test results were obtained that were not ionized and that no parent molecule peak was observed (not shown). From this result, it is considered that in the polyacrylic acid resin sheet, the presence of the side chain carboxyl group, particularly the OH group, is involved in the ionization, and the presence of the OH group is important. From these, as a resin sheet that can be used in the analysis method of the present invention, for example, a transparent nylon sheet, a polyethylene ionomer sheet, a cyclic polyolefin sheet, an acrylic sheet, an aromatic nylon sheet, a polycarbonate sheet , Polystyrene copolymer sheets, polylactic acid sheets, and the like. Among these resin sheets, resin sheets made of cyclic polyolefin, aromatic nylon, polycarbonate, and polyacrylic acid (polyacrylic acid in a broad sense including polymethacrylic acid) are preferable. Mass spectrum with less noise as a control spectrum (a spectrum performed without placing a sample as a so-called blank) having a specific absorption region near the wavelength and having a small absorption peak in other wavelength regions. This is because

  The thickness of the resin sheet is not particularly limited as long as it physically supports the analyte and can be used as a sample mounting table, and is approximately 1 to 1000 μm. However, depending on the material of the resin sheet, even a resin sheet of less than 1 μm may be used, and a resin sheet of more than 1000 μm may be used. Moreover, although it may have flexibility, if it compares, what has rigidity of the grade which can be utilized as what is called a glass slide is more preferable. As will be described below, as long as an analyte is placed on a resin sheet, it can be used for both microscopic observation and mass spectrometry.

  In addition, a single sheet may be used as the resin sheet, but a laminated sheet in which another sheet member serving as a supporting substrate is bonded from the viewpoint of further reinforcing the support of the sample may be used. In this case, the resin sheet listed above may be used for the surface on which the sample is placed, and the sheet member used as the support base may be made of a material other than the resin listed above.

  In the analysis, a flat resin sheet having no irregularities on the surface is usually used, but it does not necessarily have to be a flat sheet having no irregularities. FIG. 1 is a schematic perspective view showing an example. A resin sheet 3 in which the sample placement portions 3 as shown in FIG. 1 are arranged in a matrix can also be used. This resin sheet 1 has a large number of sample placement portions (convex shape portions) 3 that facilitate placing the analyte on one side of the sheet substrate 2, and the sample placement portions 3 extend in parallel with each other. Dimensionally arranged. The shape of the sample mounting portion 3 is not particularly limited as long as the shape is raised on the surface of the resin sheet 1, and as shown in FIG. 1, a convex portion or a horizontal portion having a substantially circular horizontal cross section like a natural mountain. Examples include a convex part (not shown) having a substantially rectangular cross section and a trapezoidal vertical cross section. In such a resin sheet 1, mass analysis can be continuously performed after a large number of analytes are previously placed on the sample placement portion 3 of the resin sheet 1. As a result, it is possible to efficiently perform microarray analysis that examines how proteins change in cells and biological tissues over time. Moreover, since the sample mounting part 3 is a convex part, there is also an advantage that the analyte can be easily irradiated with the laser. Although not shown, a resin sheet having one or a plurality of recesses for placing an analyte on a flat sheet base material may be used.

  The resin sheet is preferably transparent. When a cell or biological tissue section is used as an analyte, it is often observed with a microscope after various staining such as HE staining (hematoxylin / eosin staining). By using a transparent resin sheet, cells or the like placed or fixed on the resin sheet can be stained and observed with a microscope. In addition, it is very convenient because mass analysis can be performed immediately with the analyte after microscopic observation as it is. As long as the above object can be achieved, the transparency is not limited. For example, in the case of the above-described transparent nylon, a resin sheet having a transmittance of 80% or more in a visible region of 380 nm to 780 nm at a thickness of 1 mm is preferably used.

  The analyte is supported on such a resin sheet. This support may be merely placing the analyte, but from the viewpoint of fixing the analyte on the resin sheet and allowing the resin sheet to function as a matrix agent, the analyte is applied on the resin sheet and dried. It is desirable. As a result, the analyte can be brought into close contact with the resin sheet after fixing.

  The method of application and drying is not particularly limited. For example, a method of pressing an analyte as it is on a resin sheet and leaving it at room temperature, mixing an analyte with an appropriate solvent such as water or ethanol, applying it to the resin sheet, and evaporating the solvent. Is exemplified. The solvent used at this time is preferably a substance that does not remain in the analyte as a matrix after transpiration, or a substance that has been present in the analyte from the beginning even if it remains, such as water. Moreover, the organic solvent which does not produce the influence which is unpreferable to a resin sheet, for example, a deformation | transformation of a resin sheet, or a resin composition, for example, methanol, ethanol, acetone, etc. is illustrated.

  Immediately after placing the analyte, it is possible to proceed to the next step (2). However, the resin sheet should be sufficiently fixed to reduce the solvent used and the volatile components in the sample. Therefore, it is preferable to provide a sufficient drying / fixing time, specifically, a drying / fixing time of several hours to one day.

  The thickness of the analyte may be about 1 to 100 μm. If it exceeds 100 μm, the resin sheet may not function as a matrix material. When the thickness is less than that, it is considered that the sample is not sufficiently ionized. Of course, this numerical value is only a guideline, and is appropriately determined so that a preferable result can be obtained depending on the content of the analyte, the type and thickness of the resin sheet, and the like.

  Next, (2) the analyte of the resin sheet on which the analyte is placed is irradiated with an infrared laser and analyzed by mass spectrometry. The configuration of the mass spectrometer for performing mass spectrometry may be the same as that of a mass spectrometer used for known MADLI method analysis, and mass spectrometers having various configurations are used. For example, a single focusing magnetic field deflection mass spectrometer, a quadrupole mass spectrometer, an ion trap mass spectrometer, a time-of-flight analyzer, a double focusing mass spectrometer, and the like are exemplified. Among these, if the detection time is extended in principle, there is no upper limit to the detectable mass, and a time-of-flight analyzer (TOF) that can actually measure a wide range of masses of several hundred to several hundred thousand is possible. Preferably used.

The laser beam to be irradiated may be a laser beam having a vibration wavelength in the infrared region, and a laser beam having a wavelength of approximately 0.7 μm to 1 mm, more preferably 1 to 30 μm is used. Specifically, a CO ₂ laser (wavelength: 9.2 to 10.8 μm) or Er: YAG (wavelength: about 2.9 μm) laser used in the MALDI method, a wavelength of 5.6 to 6.0 μm is used. The laser which has is illustrated. Conditions such as laser light output and pulse time can be set as appropriate. However, the laser light source may emit light having a wavelength outside this range. For example, a device capable of changing from a nitrogen laser light source to a laser beam having an arbitrary wavelength can be used.

In the present invention, the resin sheet functions as a matrix substance as described above, and facilitates generation of analyte ions, particularly molecular ions (M ⁺ ) of proteins and peptides contained therein. As a result, the generated molecular ions are detected by mass spectrometry. Although the resin sheet is utilized to detect the molecular ion (M ⁺⁾ as a main object, the present invention is not limited only to this molecular ion (M ^+), including the molecular ion (M ⁺⁾ (M + H ) ⁺ And (M−H) ⁺ Other various fragment ions are detected, and are intended to be used for the purpose of widely collecting information on the molecular structure of the analyte.

  The above-described embodiments and the following examples are exemplifications, and the present invention is not limited to the above-described embodiments and the following examples, and the scope of the claims and equivalents thereof. All included changes are intended to be included in the present invention.

Hereinafter, the present invention will be described in more detail based on the following examples.
First, several types of resin sheets considered to be usable first were selected, and absorption spectra were measured. The result is shown in FIG. The absorption spectrum was obtained by using a resin sheet having a thickness of 150 μm, an ultraviolet visible near infrared spectrophotometer (ultraviolet / near infrared region: V-670, manufactured by JASCO Corporation) and a Fourier transform infrared spectrophotometer (infrared: FT / IR, manufactured by the same company). 2A is a transparent nylon sheet, FIG. 2B is a polyethylene ionomer sheet (Hylamine 1706: Mitsui Dupont Polychemical), and FIG. 2C is a cyclic polyolefin sheet (Apel 6013T: Mitsui Chemicals). (D) is an acrylic sheet (Delbed 560F: manufactured by Asahi Kasei Chemicals), (e) is an aromatic nylon sheet (Grivory GTR45: manufactured by Ems Chemie), and (f) is a polycarbonate sheet (Macrolon 205: Bayer). (G) is a polystyrene copolymer sheet (Clearpact TI-300: manufactured by DIC Corporation), and (g) is a polylactic acid sheet.

  FIG. 3 shows the chemical structural formulas of the resins used in the resin sheet shown in FIG. 1 (where m and n are positive integers), and Table 1 shows the characteristics of these resin sheets. Table 1 shows the effect of the solvent on the resin sheet in addition to the absorption peak wavelength and Tg (or MP) (presence / absence of denaturation of the resin sheet), and the presence / absence of dissolution that occurs when the laser of the absorption wavelength of each resin sheet is irradiated. In addition to the target molecular ion spectrum (dissolution of absorption wavelength), some of the additional spectrum generated (see Example 2 below) was also shown.

  Next, mass spectrometry was performed using angiotensin II (molecular weight: 1046), which is a peptide involved in vasoconstriction. 1 μl of a solution obtained by dissolving angiotensin II in a 0.1% TFA (trifluoroacetic acid) aqueous solution to a concentration of 100 pmol / μl was dropped onto each resin sheet (thickness 150 μm), and the solvent was evaporated under reduced pressure. . Thereafter, mass spectrometry was performed under the following measurement conditions. The result is shown in FIG. As shown in FIG. 4, a peak due to the molecular ion of angiotensin II was clearly observed at m / z = 1046 when any resin sheet was used.

Measurement conditions Laser wavelength: 5.6 to 6.0 μm
Laser energy: 600μJ
Apparatus: Applied BioSystems, Voyager DE Pro

  On the polyacrylic acid resin sheet, mass spectrometry was performed using angiotensin II as an analysis object under the above conditions. The resin sheet was produced by dropping a polyacrylic acid solution prepared at a concentration of 25% onto a flat portion of a Teflon (registered trademark) processed container and then irradiating with UV light. The obtained mass spectrum is shown in FIG.

  Mass analysis was performed on the cyclic polyolefin sheet (thickness 150 μm) under the above conditions. Sample solution (concentration: 100 pmol / μl: in addition to this, a saturated aqueous solution of CsI for calibration) Tuftsin (molecular weight: 50.6), a peptide represented by Lys-Thr-Lys-Pro-Arg, dissolved in ultrapure water 1 μl) was dripped onto the resin sheet. Mass spectrometry was performed on a sample in which the solvent was evaporated by evacuation immediately after dropping of the sample solution and a sample that had passed 24 hours after dropping. The results are shown in FIGS. 6 (a) and 6 (b).

  In all cases, the peak considered to be due to the Tuftsin molecular ion was clearly observed, and almost no other fragment ions were observed. Further, when measured after 24 hours, the peak height of the molecular ion was higher than that immediately after that, the CsI peak for calibration was clearly observed, and the noise was reduced.

  Further, mass spectrometry was performed on the cyclic polyolefin sheet (thickness 150 μm) under the above measurement conditions. For mass spectrometry, 1 μl of a sample solution (concentration: 100 pmol / μl each, including a saturated aqueous solution of CsI for calibration) added dropwise to Tuftsin and succinic acid (molecular weight: 500. 6) in ultrapure water. Then, immediately after the dropping, a vacuum was drawn to evaporate the solvent. The result is shown in FIG. For reference, FIG. 8 shows the measurement results when the sample solution was directly dropped onto the sample stage of the mass spectrometer without using a resin sheet.

  According to this, when a resin sheet was used, a clear peak of m / z = 501 indicating the parent ion of Tuftsin was observed, and no interference with succinic acid was observed. On the other hand, when no resin sheet was used, a peak at m / z = 501 indicating the parent ion of Tuftsin was not observed as shown in FIG.

  As described above, according to the present invention, by placing or applying the analyte on the resin sheet, it is possible to analyze a high-molecular substance such as protein or peptide by mass spectrometry without mixing with the matrix substance. As a result, mass spectrometry can be efficiently performed in fields such as proteomics, metabolomics, and biological imaging, and it is thought that this will greatly contribute to the elucidation of new biological functions and the efficiency of clinical tests.

DESCRIPTION OF SYMBOLS 1 Resin sheet 2 Sheet base material 3 Sample mounting part (convex shape part)

Claims (9)

A method for analyzing an analyte by laser desorption ionization mass spectrometry,
(1) placing the analyte on a resin sheet;
(2) a step of analyzing the analyte by irradiating the analyte placed on the resin sheet with an infrared laser, ionizing the analyte and detecting by mass spectrometry;
Analytical methods including:   The analysis method according to claim 1, wherein the resin sheet is a resin sheet selected from cyclic polyolefin, aromatic nylon, polycarbonate, and polyacrylic acid.   The analysis method according to claim 1 or 2, wherein a thickness of the resin sheet is 1 µm or more and 1000 µm or less. The resin sheet has a plurality of convex portions formed on at least one side,
The analysis method according to claim 1, wherein the convex portions are arranged so as to extend in parallel to each other.   The analysis method according to any one of claims 1 to 4, wherein the resin sheet is involved in the generation of molecular ions of an analyte as a matrix substance and enables detection of the molecular ions. .   The analysis method according to any one of claims 1 to 5, wherein the analyte includes one or more of proteins, polypeptides, peptides, biological tissue sections or cells containing the components.   The analysis method according to any one of claims 1 to 6, wherein the resin sheet on which the peptide is placed is irradiated with laser light having a wavelength of 5.6 to 6.0 µm. A method of using a resin sheet in a method of analyzing an analyte by laser desorption ionization mass spectrometry,
A method of using a resin sheet for detecting parent ions generated by placing an analyte on a resin sheet and irradiating the analyte placed on the resin sheet with an infrared laser.   The method for using a resin sheet according to claim 7, wherein the resin sheet is a resin sheet selected from cyclic polyolefin, aromatic nylon, polycarbonate, and polyacrylic acid.

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