JP6148540B2 - Method for quantitative analysis of granulin peptide using mass spectrometer and program for analysis - Google Patents

Description

The present invention, the content of grayed La New phosphopeptide of biological samples such as blood, to the analysis method of quantitatively individually with mass spectrometer. Furthermore, the present invention relates to a program for the analysis and a mass spectrometer.

  Progranulin (PGRN, also known as: Proepithelin / PCDGF / PEPI / GEP / GP88) is a precursor protein with a granulin (GRanulin, GRN, also known as: Epithelin) domain, PGRN is blood It is known that it undergoes processing by a proteolytic enzyme (for example, neutrophil elastase) in the middle to produce 7 types of GRN peptides, and Fig. 1 is an explanatory diagram showing the relationship between PGRN and GRN peptides. The numbers in the middle represent the number of amino acid residues from the N-terminus of PGRN including the signal peptide, with the upper row corresponding to the mouse PGRN sequence and the lower row to the human PGRN sequence.

  Mouse PGRN is a protein of about 63 kDa consisting of a total length of 589 amino acids (SEQ ID NO: 1). Of 7 types of granulin peptides, GRN3 peptide (SEQ ID NO: 2) consisting of 56 amino acids and 55 A GRN4 peptide (SEQ ID NO: 3) consisting of amino acids has been identified (Non-patent Document 1). Human PGRN is a protein of about 63 kDa consisting of a total length of 592 amino acids (SEQ ID NO: 4), a GRN3 peptide (SEQ ID NO: 5) consisting of 56 amino acids, and a GRN4 peptide (SEQ ID NO: 6) consisting of 55 amino acids. ) Has been identified.

  PGRN is known to be a protein related to various diseases. For example, in the case of insulin resistance, which is a pathological condition characteristic of type 2 diabetes, it has been reported that PGRN increases in blood and induces insulin resistance (Non-patent Document 2). In recent years, it has been reported that GRN peptides produced by PGRN processing and the like are involved in the onset of rheumatism (Non-patent Document 3). Thus, since PGRN and GRN peptides are associated with various diseases, development of screening for drugs targeting PGRN as a drug discovery target and development of diagnostic methods such as rheumatism and diabetes are expected. . For example, Patent Document 1 proposes using PGRN and GRN peptides as insulin resistance markers. Quantitative techniques for PGRN and GRN peptides are considered useful for detection of various diseases and elucidation of disease states.

JP 2009-204475 A

Bateman A. et al., J. Endocrinol, vol. 158, pp.145-151 (1998) Matsubara T. et al., Cell Metab., Vol. 15, pp. 38-59 (2012) Tang W. et. Al., Science, vol. 332, pp. 478-484, (2011)

  Since PGRN is processed extracellularly to produce GRN peptides, PGRN and GRN peptides in biological samples such as blood must be individually quantified in order to detect diseases and elucidate disease states. However, conventionally, the enzyme antibody method used for quantification of PGRN and GRN peptides cannot individually and selectively detect PGRN and GRN peptides in extracellular biological samples.

  LC / MS / in combination with liquid chromatography (LC) and tandem mass spectrometer (MS / MS) as a method to individually quantify proteins and peptides in samples containing various contaminants such as biological samples Applications of multiple reaction monitoring (MRM) analysis using MS are progressing. In MRM analysis, each component in a sample is temporally separated by LC and introduced into a mass spectrometer. The sample introduced into the mass spectrometer is ionized by an ionization probe to generate various ions. The ions generated by the probe (precursor ions) are selected in the first mass separation section in the previous stage, and specific ions to be detected are selected based on the mass-to-charge ratio (m / z), and collision-induced dissociation is performed at the ion cleavage section. Cleaved into multiple types of ions (product ions) by (CID). A specific product ion is transmitted to the detector based on m / z in the second mass separation unit at the subsequent stage, and the product ion is quantified by the detector. In MRM analysis, for each component to be analyzed, the m / z of one or more precursor ions selected by the first mass separation unit, and one or more types selected by the second mass separation unit for each precursor ion It is necessary to determine m / z of the product ion in advance.

  Since proteins and peptides have a high molecular weight, in MRM analysis, analysis is performed on peptide fragments obtained by cleaving a protein in a sample at a specific amino acid sequence position by protease treatment to reduce the molecular weight. In MRM analysis of samples containing various contaminants, proteins and peptides other than the analysis target may generate the same precursor ions and product ions as the analysis target protein and peptide. Therefore, in MRM analysis of proteins and peptides in biological samples, it is important to appropriately select a combination of precursor ions and product ions (MRM conditions) specific to the protein or peptide to be analyzed.

  In addition, in order to individually quantify a plurality of types of proteins and peptides contained in one sample, it is necessary to select a combination of precursor ions and product ions specific to each analysis target. However, since PGRN is a precursor of GRN peptide, both generate common precursor ions and product ions. That is, since the amino acid sequence of the GRN peptide is in common with a part of the amino acid sequence of PGRN, if a biological sample in which PGRN and GRN peptide are mixed is fragmented with a protease, they can produce the same fragment. For this reason, it is generally difficult to select a combination of precursor ions and product ions (MRM conditions) specific to each of the PGRN and GRN peptides.

  Of the seven types of GRN peptides, only two types of GRN3 and GRN4 have been identified as cleavage sites from PGRN, and other GRN cleavage sites have not been identified at this stage. Furthermore, the sugar chain modification structures of PGRN and GRN peptides still have many unexplained parts. Therefore, the m / z value predicted from the amino acid sequence does not always match the m / z value of the precursor ion or product ion actually obtained, and identification is not easy. Under such circumstances, there is no example of individual quantification of GRN peptide and its precursor PGRN in a biological sample, and a method for individually quantifying PGRN and GRN in an extracellular biological sample such as blood. Is not established.

In view of the above situation, the present invention aims to provide a quantitative analytical method for quantifying individual G RN peptide biological sample such as blood.

The present invention, the content of grayed La New phosphopeptide derived from a living organism in a sample, on the analysis method of quantifying using mass spectrometer. Examples of biological samples include blood, serum, tissue extract, cell extract, urine, cerebrospinal fluid and the like. These biological samples are, for example, samples derived from humans and mice.

  The analysis method of the present invention comprises a step of preparing a biological sample containing the protein and / or peptide to be analyzed; the protein and peptide in the sample are selectively cleaved at a predetermined amino acid position, A step of obtaining a peptide fragment; a sample after cleavage treatment is analyzed using a mass spectrometer, and two or more predetermined product ions are detected and quantified; and based on the detected amount of each product ion, Calculating the content of the predetermined peptide fragment in the sample.

  In order to selectively cleave proteins and peptides in a sample at a predetermined amino acid position, treatment using a protease such as trypsin is preferable. In one embodiment, the cleavage treatment is treatment in which peptide bonds on the C-terminal side of lysine residues and arginine residues are selectively cleaved.

  The analysis of the sample after the cutting treatment is performed by a mass spectrometer including a first mass separation unit, an ion cleavage unit, a second mass separation unit, and a detector. For example, a triple quadrupole mass spectrometer is used as the mass spectrometer. Since a biological sample contains various contaminants, each component in the sample may be temporally separated by liquid chromatography (LC) before the sample is introduced into the mass spectrometer. preferable. That is, in the analysis method of the present invention, LC / MS / MS combining a liquid chromatography (LC) and a tandem mass spectrometer (MS / MS) is preferably used.

  Operations of the first mass separation unit, the ion cleavage unit, the second mass separation unit, and the detector of the mass spectrometer can be controlled by the control unit. The control unit includes a computer that performs the analysis and analyzes the obtained data.

  After the sample introduced into the mass spectrometer is ionized by the ionization probe, ions having a specific mass-to-charge ratio are selected as precursor ions in the first mass separation unit. Each of the selected precursor ions is cleaved into a plurality of types of product ions at the ion cleavage portion. Product ions are selected based on the mass-to-charge ratio in the second mass separation unit, and product ions having a specific mass-to-charge ratio are detected and quantified by the detection unit.

  The mass-to-charge ratio of precursor ions selected by the first mass separation unit and the mass-to-charge ratio of precursor ions selected by the second mass separation unit both correspond to the type of peptide fragment to be quantified, It is predetermined. In the mass spectrometer, (A) the amino acid sequence of the granulin peptide consists of at least 6 consecutive amino acids, and neither the N-terminal sequence portion nor the C-terminal sequence amino acid sequence portion of the amino acid sequence of the granulin peptide is included. , A granulin-intermediate sequence fragment, and (B1) a granulin-N-terminal addition fragment in which three or more amino acids are further linked to the N-terminus of at least three amino acid sequences continuous from the N-terminal side of the amino acid sequence of the granulin peptide And (B2) a granulin-C-terminal addition fragment in which three or more amino acids are further bonded to the C-terminus of at least three amino acid sequences continuous from the C-terminal side of the amino acid sequence of the granulin peptide. One or more end-added fura selected Instrument; including, product ions generated by the dissociation of the precursor ions from each of the two or more peptide fragments are quantified.

  The amount of each fragment is related to the calibration curve obtained in advance based on the amount of product ions obtained by mass spectrometry, or the peak area of product ions derived from the internal standard sample added to the sample. Etc. The content of granulin peptide is calculated based on the difference between the amount of granulin-intermediate sequence fragment and the amount of terminal addition fragment.

  In one embodiment of the present invention, granulin peptides to be quantified are granulin 3 peptide and granulin 4 peptide. In this case, in the mass spectrometer, the product ion generated by cleavage of the precursor ion derived from each of the granulin 3-intermediate fragment and the granulin 4-intermediate sequence fragment, and the granulin 3-N-terminal addition fragment and the granulin 4-C-terminal are obtained. The amount of the three or more peptide fragments including the product ion generated by the cleavage of one or more terminal addition fragments selected from the group consisting of the addition fragments is calculated. The content of granulin 3 peptide is calculated based on the difference between the amount of granulin 3 -intermediate sequence fragment and the amount of terminal addition fragment. The content of granulin 4 peptide is calculated based on the difference between the amount of granulin 4 -intermediate sequence fragment and the amount of terminal addition fragment.

  From the viewpoint of accuracy of quantification, it is preferable that both the amount of granulin 3-N-terminal addition fragment and the amount of granulin 4-C-terminal addition fragment are quantified. In this case, the content of granulin 3 peptide is calculated based on the difference between the amount of granulin 3-intermediate sequence fragment and the amount of granulin 3-N-terminal addition fragment. The content of granulin 4 peptide is calculated based on the difference between the amount of granulin 4-intermediate sequence fragment and the amount of granulin 4-C-terminal addition fragment.

  Furthermore, the present invention relates to a program for causing a computer in a control unit of the mass spectrometer to execute the analysis, and a mass spectrometer including a computer in which the program is installed.

According to the present invention, the GRN peptide in a biological sample can be individually quantified. Therefore, it is expected to be applied to examinations for detection of various diseases related to PGRN and GRN peptides, research for elucidating the pathology of these diseases, and the like.

It is a figure for demonstrating the relationship between progranulin (PGRN) and a granulin (GRN) peptide. It is a schematic diagram for demonstrating fragmentation of PGRN and GRN by protease. It is a figure which shows an example of the cleavage position by the protease in the GRN3 peptide of mouse | mouth PGRN, and the amino acid sequence of the vicinity. It is a figure which shows an example of the cleavage position by the protease in the GRN4 peptide of mouse | mouth PGRN, and the amino acid sequence of the vicinity. It is a figure which shows an example of the cleavage position by the protease in the GRN3 peptide of human PGRN, and the amino acid sequence of the vicinity. It is a figure which shows an example of the cleavage position by the protease in the GRN4 peptide of human PGRN, and the amino acid sequence of the vicinity. It is a flowchart which shows an example of the analysis method of this invention. It is a block diagram of a tandem type mass spectrometer. It is a chromatogram showing the precursor scan result of purified mouse progranulin. It is a MS spectrum showing the precursor scan result of the fragment 1 (sequence number 7). It is a MS spectrum showing the precursor scan result of the fragment 2 (sequence number 8). It is a MS spectrum showing the precursor scan result of the fragment 3 (sequence number 9). It is a MS spectrum showing the precursor scan result of the fragment 4 (sequence number 10). It is a MS spectrum showing the product ion scan result of the fragment 1 (sequence number 7). It is a MS spectrum showing the product ion scan result of the fragment 2 (sequence number 8). It is a MS spectrum showing the product ion scan result of the fragment 3 (sequence number 9). It is a MS spectrum showing the product ion scan result of the fragment 4 (sequence number 10). 2 is a calibration curve obtained from the peptide content of fragment 1 and the amount of product ions, and an MRM chromatogram of product ions. 2 is a calibration curve obtained from the peptide content of fragment 2 and the amount of product ions, and an MRM chromatogram of product ions. It is a calibration curve obtained from the peptide content of fragment 3 and the product ion content, and an MRM chromatogram of product ions. 5 is a calibration curve obtained from the peptide content of fragment 4 and the product ion content, and an MRM chromatogram of product ions. It is a chromatogram showing the spike test result to human serum of fragment 1. It is a chromatogram showing the spike test result to human serum of fragment 2. It is a chromatogram showing the spike test result to human serum of Fragment 3. It is a chromatogram showing the spike test result to human serum of Fragment 4. It is a chromatogram of each product ion when MRM analysis of mouse serum is performed by the analysis method of the present invention. It is a graph showing the GRN peptide density | concentration calculated | required from (A) each fragment density | concentration and (B) fragment density | concentration at the time of performing MRM analysis of mouse | mouth serum by the analysis method of this invention.

The analysis method of the present invention comprises a step of preparing a biological sample containing the protein and / or peptide to be analyzed; the protein and peptide in the sample are selectively cleaved at a predetermined amino acid position, A step of obtaining a peptide fragment; a step of analyzing a cleaved sample by a mass spectrometer and detecting and quantifying two or more kinds of predetermined product ions; and the sample based on the detected amount of each product ion Calculating the content of the predetermined peptide fragment therein.
First, the principle by which progranulin (PGRN) and granulin (GRN) peptides are individually quantified according to the present invention will be described.

  FIG. 2 is a schematic diagram for explaining the fragmentation of PGRN and GRN by protease, with the upper row representing the GRN peptide and the lower row representing PGRN. In the illustrated example, the GRN peptide is cleaved at predetermined cleavage sites I to III by protease treatment and fragmented into four types of fragments (GRN-a, GRN-b, GRN-c, and GRN-d). PGRN is treated by protease treatment with cleavage sites I to III similar to GRN, cleavage site X further N-terminal than the N-terminus of the GRN peptide sequence, and C-terminal further than the C-terminus of the GRN peptide sequence. It is cleaved at the cleavage site Y and fragmented into four types of fragments (N-Linker + GRN, GRN-b, GRN-c, and GRN + C-Linker).

  When a sample containing both GRN and PGRN is treated with protease, a peptide fragment containing neither the N-terminus nor the C-terminus of the amino acid sequence of the GRN peptide (hereinafter referred to as “GRN-”), such as the GRN-b fragment or the GRN-c fragment. (Sometimes referred to as “intermediate sequence fragments”) are generated from both GRN and PGRN. Therefore, if the content of the GRN-intermediate sequence fragment in the sample after protease treatment is quantified, the total amount of GRN and PGRN can be obtained.

  The GRN N-terminal fragment (GRN-a) obtained by protease treatment of GRN and the C-terminal fragment (GRN-d) are obtained by fragmentation of PGRN (N-Linker + GRN and GRN + C-Linker) and Is different. The N-Linker + GRN fragment is obtained by adding a sequence consisting of a plurality of amino acids to the N-terminal side of the N-terminal fragment (GRN-a) obtained by GRN fragmentation (hereinafter referred to as “GRN-N terminal addition”). Sometimes referred to as "fragment"). The GRN + C-Linker fragment is a C-terminal fragment (GRN-d) obtained by fragmentation of GRN, to which a sequence consisting of a plurality of amino acids is further added (hereinafter referred to as “GRN-C-terminal addition”). Sometimes referred to as "fragment").

  These GRN-N end addition fragments and GRN-C end addition fragments (hereinafter sometimes collectively referred to as “end addition fragments”) do not originate from the GRN peptide and are unique to PGRN. When product ion analysis by MS / MS is performed using ions derived from end-added fragments as precursor ions, product ions containing amino acid sequences not included in GRN can be obtained. Therefore, these end-added fragments can be individually quantified by MS / MS, distinguishing them from N-terminal fragments (GRN-a) derived from GRN peptides and C-terminal fragments (GRN-d) derived from GRN peptides. .

  As described above, the total content of GRN and PGRN is determined from the content of the intermediate sequence fragment, and the content of PGRN is determined from the content of the terminal addition fragment. The content of the GRN peptide is determined by calculating the difference between the two.

  As described above, in MRM analysis of biological samples, proteins and peptides other than the analysis target may generate the same precursor ions and product ions as the analysis target. It is important to appropriately select the combination (MRM condition). In order to ensure the specificity of the precursor ion, it is preferable to select a peptide comprising 6 or more amino acid residues as the intermediate sequence fragment and the terminal addition fragment to be analyzed. The upper limit of the peptide fragment length is not particularly limited, but is preferably 35 amino acid residues or less from the viewpoint of ease of ionization. The number of amino acid residues of the fragment to be analyzed is preferably about 8 to 25, and more preferably about 10 to 20.

  Further, from the viewpoint of giving the product ion specificity, the N-terminal addition fragment to be analyzed is preferably one in which 3 or more amino acids are further bonded to the N-terminus of the amino acid sequence of the granulin peptide. Similarly, the C-terminal addition fragment to be analyzed is preferably one in which 3 or more amino acids are further bonded to the C-terminus of the amino acid sequence of the granulin peptide.

  When selecting a pair of precursor ion and product ion in MRM analysis, as described above, it is possible to select a precursor ion based on a highly specific amino acid sequence, and also to select a product ion with good quantitative and reproducibility. preferable.

  In quantitative analysis using a mass spectrometer, if the peptide sequence is unknown, it is difficult to identify ions based on the mass-to-charge ratio (m / z). Therefore, at the present stage, of the seven types of GRN peptides, two types of GRN3 and GRN4 whose cleavage positions from PGRN are specified can be quantified by the above method. In the future, when the sequence of other GRN is clarified, other GRN peptides can be quantified from the difference between the amount of GRN-intermediate sequence fragment and the amount of terminal addition fragment according to the same principle.

Hereinafter, the analysis method of the present invention will be described in more detail with reference to the steps shown in the flowchart of FIG.
[Sample preparation]
First, a biological sample to be analyzed is prepared (sample preparation step S100). Samples to be analyzed in the present invention are biological samples such as blood, serum, plasma, tissue extract, cell extract, urine, and cerebrospinal fluid. These samples contain progranulin (PGRN) and / or granulin (GRN) peptide. As mentioned above, PGRN can be processed by proteases in the blood to yield GRN peptides. In the present invention, PGRN and GRN (particularly GRN3 and GRN4) in a sample are quantified.

[Sample preparation]
(Selective fragmentation by protease)
Proteins and peptides in the sample are selectively cleaved at predetermined sequence positions to obtain a plurality of peptide fragments (step S202). Protease treatment is preferably performed to selectively cleave proteins and peptides at predetermined sequence positions. Usually, proteases recognize amino acid sequences and selectively cleave specific bonds of specific sequences.

  Examples of proteases include trypsin (which cleaves peptides at the C-terminal side of basic amino acid residues (Arg and Lys)), lysyl endopeptidase (which cleaves peptides at the C-terminal side of Lys residues), arginine endopeptidase ( Cleave the peptide on the C-terminal side of Arg residues), chymotrypsin (cleave the peptide on the C-terminal side of aromatic amino acid residues (Phe, Tyr and Trp)), pepsin (aromatic residues (Phe, Tyr and Crp is cut at the N-terminal side of Trp).

  In the present invention, the cleavage position by the protease is determined so as to obtain a fragment that produces a combination of a precursor ion and a product ion peculiar to PGRN or GRN to be analyzed. Precursor ions and products necessary for individual quantitative analysis of PGRN, GRN3, and GRN4, as shown in the examples below, by selectively cleaving peptide bonds on the C-terminal side of lysine residues and arginine residues. It has been confirmed that a set of ions can be obtained. Therefore, trypsin is preferably used as the protease.

  In addition, protease can also be used in combination of 2 or more types. For example, when lysyl endopeptidase and arginine endopeptidase are used in combination, peptide bonds on the C-terminal side of lysine residues and arginine residues can be selectively cleaved. Further, trypsin and lysyl endopeptidase and / or arginine endopeptidase may be used in combination in order to make the selective product cleavage by protease more complete.

  The conditions for the protease treatment are not particularly limited, and an appropriate protocol corresponding to the protease used is employed. For example, it is preferable to incubate at a temperature of about 37 ° C. for about 4 to 20 hours in a buffer solution adjusted to near the optimum pH of the protease.

(Specific examples of selective fragmentation)
FIG. 3A and FIG. 3B show the cleavage positions of the mouse GRN3 peptide and its neighboring amino acid sequences, and the GRN4 peptide and its neighboring amino acid sequences by trypsin digestion.

  In FIG. 3A, fragment 1 (198-211, SEQ ID NO: 7) is an additional 7 amino acids (AVSLPFS) at the N-terminus of a 7 amino acid sequence (VVCPDAK) continuous from the N-terminal side of the amino acid sequence of granulin 3 peptide. Is a granulin 3-N-terminal addition fragment to which Fragment 2 (212-227, SEQ ID NO: 8) consists of 16 consecutive amino acids (TQCPDSTCCELPTTGK) of the amino acid sequence of granulin 3 peptide, and the N-terminal sequence portion and the C-terminal sequence amino acid sequence portion of the amino acid sequence of granulin 3 peptide Is a granulin 3-intermediate sequence fragment.

In FIG. 3B, fragment 3 (297-309, SEQ ID NO: 9) consists of 13 consecutive amino acids (LNTGAWGGCCPFAK) in the amino acid sequence of granulin 4 peptide, and the N-terminal sequence portion and C-terminal of the amino acid sequence of granulin 4 peptide. It is a granulin 4-intermediate sequence fragment that does not contain any of the sequence amino acid sequence parts. Fragment 4 (331-344, SEQ ID NO: 10) is a granulin in which 10 amino acids (MGILQVPWMK) are further bound to the C-terminus of the four amino acid sequences (GTCE) continuous from the C-terminal side of the amino acid sequence of granulin 4 peptide. 4-C terminal addition fragment.
In a later example, an example in which GRN3, GRN4, and PGRN are individually quantified from the amounts of these four fragments will be described.

  The cleavage positions of human GRN3 peptide and GRN peptide by trypsin digestion are shown in FIGS. 4A and 4B. In each of these drawings, the region surrounded by a rectangle is granulin peptide. The granulin 3-N end addition fragment (199-212, SEQ ID NO: 11) in FIG. 4A and the granulin 4-C end addition fragment (333-347, SEQ ID NO: 12) in FIG. 4B are the mouse granulin 3-N, respectively. The homology of the end addition fragment and granulin 4-C end addition fragment is high, and the number of amino acid residues of the fragment is also approximately the same. Therefore, it can be understood that these end-added fragments are suitable as MRM analytes for quantification of PGRN and GRN in human biological samples.

(Pre-fragmentation processing)
In general, prior to protease treatment, denaturation treatment and alkylation treatment of proteins and peptides in a sample are performed (step S201). The conditions for the modification treatment and the alkylation treatment are not particularly limited, and known conditions are appropriately employed. In addition, when the amount of fragments is calculated by associating the amount of product ions obtained by mass spectrometry with the peak area of product ions derived from the internal standard sample, an internal standard sample with a known concentration is added prior to protease treatment. It is preferable.

(Post-fragmentation processing)
The sample after the protease treatment may be subjected to treatments such as desalting, solubilization, concentration, and drying as necessary. For example, desalting and concentration can be performed using a spin column for solid phase extraction or the like. (Step S203)

[Analysis with mass spectrometer]
The sample after fragmentation with protease is analyzed using a mass spectrometer. Since a biological sample contains various contaminants, each component in the sample may be temporally separated by liquid chromatography (LC) before the sample is introduced into the mass spectrometer. Preferred (LC analysis S301). That is, the analysis method of the present invention is preferably performed using LC / MS / MS in which liquid chromatography (LC) and a tandem mass spectrometer (MS / MS) are combined. In LC / MS / MS, each component in a sample is temporally separated by LC before mass spectrometry. Therefore, the product ion derived from the fragment to be analyzed can be detected with high accuracy and high sensitivity by switching the combination of the mass-to-charge ratio of the predetermined precursor ion and product ion according to the retention time of each component, so the accuracy of quantitative analysis is improved. Enhanced.

  FIG. 6 is a diagram schematically showing a configuration example of a tandem (MS / MS type) mass spectrometer used in the present invention. This mass spectrometer includes an ion source 2 for ionizing a sample to be analyzed in an analysis chamber 30 that is evacuated by a vacuum pump (not shown), and three stages of quadruples each consisting of four rod electrodes. A triple quadrupole mass spectrometer including poles 3, 4, and 5 and a detector 6 that detects ions and outputs a detection signal corresponding to the amount of ions. The first stage quadrupole 3 and the third stage quadrupole 5 are quadrupole mass filters. The second stage quadrupole 4 is merely a quadrupole (or multipole) ion guide and does not have a function of mass separation.

  A voltage obtained by synthesizing a DC voltage and a high-frequency voltage is applied to the first stage quadrupole 3, and a specific mass-to-charge ratio among various ions generated by the ion source 2 by the action of an electric field generated thereby. Are selected as precursor ions (first mass separation: precursor ion selection step S303).

  The second stage quadrupole 4 is housed in a collision cell 9 having a high hermeticity. A CID gas such as argon is introduced into the collision cell 9. Precursor ions sent from the first-stage quadrupole 3 to the second-stage quadrupole 4 collide with the CID gas in the collision cell 9 and are cleaved by collision-induced dissociation to generate product ions (ion ions). Cleavage step S304). Since this mode of cleavage is various, usually, a plurality of types of product ions having different mass-to-charge ratios are generated from one type of precursor ion. These various product ions exit the collision cell 9 and are introduced into the third stage quadrupole 5. Usually, only the high-frequency voltage is applied to the second-stage quadrupole 4, or a voltage obtained by adding a DC bias voltage to the high-frequency voltage is applied, and the second-stage quadrupole 4 converges ions. It functions as an ion guide that is transported to the subsequent third-stage quadrupole 5.

  Similarly to the first-stage quadrupole 3, a voltage obtained by synthesizing a DC voltage and a high-frequency voltage is applied to the third-stage quadrupole 5. Due to the action of the electric field generated thereby, only the product ions having a specific mass-to-charge ratio are sorted and transmitted to the detector 6 in the third-stage quadrupole 5 (product ion sorting step S305). By appropriately changing the DC voltage and the high-frequency voltage applied to the third stage quadrupole 5, the mass-to-charge ratio of ions that can pass through the third stage quadrupole 5 can be scanned (product ion scan).

  Product ions selected by the third-stage quadrupole 5 are detected by a detector, and an electrical signal corresponding to the detected amount of product ions is transmitted to the data processing unit 40. The data processing unit 40 can create a mass spectrum (MS / MS spectrum) of product ions, and the amount of product ions is quantified from the peak area of product ions (product ion detection / quantification step S306).

  The first stage quadrupole 3, the second stage quadrupole 4 and the collision cell 9, the third stage quadrupole 5, the detector 6, and the data processing unit 40 are controlled by a control unit 50 including a computer. Works below. The control unit 50 is associated with a storage unit 60 that stores a control program. When the CPU executes the program stored in the storage unit 60, the control unit 50 causes the first stage quadrupole 3, the second stage quadrupole 4 and the collision cell 9, the third stage quadrupole 5, and Each of the detector 6 and the data processing unit 40 is caused to function as a first mass separation unit, an ion cleavage unit, a second mass separation unit, and a detection unit.

  The program for performing the above analysis includes information on a fragment to be detected. The fragment information includes the mass-to-charge ratio of the precursor ion and the mass-to-charge ratio of the product ion for each of the fragments to be detected. Furthermore, when liquid chromatography (LC) is connected to the front stage of the mass spectrometer, it is preferable that the fragment information includes a retention time. In addition to the mass-to-charge ratio and the retention time, the fragment information may include a sample name, a parameter for quantification (for example, a regression equation of a calibration curve, association data such as a concentration of an internal standard), and the like. These pieces of fragment information may be stored in the storage unit 60 as an MRM condition table 61 as shown in FIG.

  The program may be provided separately from the mass spectrometer, or may be provided in a state of being stored in a storage medium, or may be provided via communication means such as an Internet line. You may complete the program for performing the said analysis by adding the information regarding the fragment used as a detection target to the existing control program for mass spectrometry. In addition, when the user inputs information about a fragment to be detected from the operation unit 51, a program for performing the analysis can be completed. Information input by the user may be a method of manually selecting information on a fragment to be detected from a keyboard or the like, or a method of selecting from information displayed on the screen of the display unit 52. Furthermore, when the user inputs a part of the fragment information to be detected (for example, a fragment name, an amino acid sequence, etc.), the control unit 50 refers to a database prepared in advance and determines other fragment information (for example, Mass-to-charge ratio and retention time) may be obtained.

  In accordance with the instruction from the analysis program, the control unit 50 determines the mass-to-charge ratio of the precursor ions selected by the first stage quadrupole 3 and the mass-to-charge ratio of the product ions selected by the third stage quadrupole 5. Switch. Depending on the LC retention time, the mass-to-charge ratio of the precursor ions and the mass-to-charge ratio of the product ions selected by the third-stage quadrupole 5 are switched, so that the product ions derived from the fragments to be detected are , Selectively reaches the detector 6 and is quantified.

[Calculation of content]
Based on the amount of product ions quantified by the mass spectrometer, the content (concentration) of PGRN and GRN peptide in the biological sample is calculated. First, the content of each fragment to be detected is calculated based on the product ion amount (steps S401 and S402). The product ion amount and the fragment amount are associated in advance by a predetermined parameter.

  As a method for associating the two, for example, a method using a calibration curve (calibration curve) by an external standard can be mentioned. For the calibration curve, analyze peptide fragments (external standard) with known concentrations under the same conditions as this analysis (analysis of biological samples), and plot the concentration and peak area (or peak intensity) of product ions. Obtained by.

Moreover, an internal standard is added to a biological sample to be analyzed in advance for measurement, and the product ion amount derived from the internal standard and the product ion amount derived from the fragment to be quantified can be correlated. The association between the two is performed by, for example, the peak area ratio of product ions. As an internal standard, for example, a heavy peptide having the same amino acid sequence as the fragment to be analyzed and having some or all of carbon and / or nitrogen substituted with heavy atoms ( ¹³ C or ¹⁵ N) is used. . Fragments derived from such heavy peptides have the same digestion rate by protease and ionization rate in mass spectrometry as proteins and peptides in biological samples, but the mass-to-charge ratio of product ions and precursor ions is derived from living organisms. Greater than that of the sample. Since the relative ratio of the fragment derived from the biological sample and the fragment derived from the internal standard of the heavy peptide is always the same, and both can be individually quantified, the concentration of the internal standard and the peak area ratio of the two can The fragment concentration can be calculated.

  Based on the concentrations of the terminal addition fragment and the intermediate sequence fragment to be analyzed, the concentrations of PGRN and GRN peptide are calculated (step S403). As mentioned above, the concentration of GRN peptide is equal to the difference between the intermediate sequence fragment concentration and the end addition fragment concentration. Also, the PGRN concentration is equal to the end addition fragment concentration.

  When calculating the concentrations of both GRN3 and GRN4, the concentration of GRN3 peptide is calculated from the difference between the concentration of GRN3-intermediate sequence fragment and the concentration of GRN3-N-terminal addition fragment, and the concentration of GRN4-intermediate sequence fragment and GRN4-C-terminal addition fragment It is preferable to calculate the concentration of GRN4 peptide from the concentration difference. When PGRN is cleaved homozygously to produce 1 to 7 GRN peptides one molecule at a time, the GRN3-N-terminal addition fragment concentration and the GRN4-C-terminal addition fragment concentration are equal. On the other hand, when PGRN is cleaved heterogeneously, the concentrations of GRN-3 peptide and GRN-4 in the sample are different, and the concentrations of GRN3-N-terminal addition fragment and GRN4-C-terminal addition fragment may be different. Even in this case, according to the above method, the concentration of each GRN can be accurately calculated.

  Based on the data obtained by mass spectrometry, the user calculates the fragment content based on the product ion content (steps S401 and 402), and calculates the PGRN content and the GRN peptide content from each fragment content ( Step S403) can be performed. These calculations may be automatically performed according to instructions from the program. For example, as described above, if the fragment information in the MRM condition table includes association data such as the regression equation of the calibration curve and the concentration of the internal standard, the CPU executes the program stored in the storage unit 60. Thus, the data processing unit 40 performs the above calculation. The obtained result can be displayed on the display unit 52 or stored in the storage unit 60 according to an instruction from the program.

  In the following, experimental examples and examples relating to the quantification of PGRN, GRN3 peptide, and GRN4 peptide in mouse serum are shown, and the present invention will be specifically described. However, the present invention is not limited to the following examples. Absent. In the following, “%” represents “% by weight” unless otherwise specified.

[Preparation of measurement sample]
(Modification and reductive alkylation treatment)
A sample of 20 μL was dispensed into a 1.5 mL Eppendorf tube, and a reducing alkylation buffer (50 mM Tris-HCl (pH 8.8, 8M urea) was added to a total of 100 μL and stirred. 10 μL of reducing agent solution (1M dithiothreitol) was added and stirred, and incubated for 60 minutes at 56 ° C. After returning the solution to room temperature, 30 μL of alkyl agent solution (0.5 M iodoacetamide) was added and stirred. Incubated for 45 minutes in the dark at room temperature.

(Trypsin digestion)
Enzyme digestion buffer (50 mM Tris-HCl, 1 mM CaCl ₂ (pH 7.6)) was added to the above solution so that the total amount was 900 μL. A trypsin solution (manufactured by Promega, Trypsin GOLD, mass spectrometry grade) was added to this solution so that the ratio of protein to trypsin (weight ratio) in the sample was 1: 100, and then at 37 ° C. for 16 hours. Incubated above.

(Desalination and concentration treatment)
A 10% formic acid aqueous solution was added to the solution after the fragmentation treatment so that the final concentration of formic acid was about 0.1% and stirred. The sample was subjected to solid phase extraction using a spin column (manufactured by GL Sciences, MonoSpin C18), and dried using a centrifugal concentrator. The obtained dry sample was stored frozen at −80 ° C. until measurement.

[Analysis by LC / MS / MS]
The dried sample was redissolved with 20 μL of mobile phase solvent (5 mM ammonium formate + 0.1% formic acid aqueous solution). The sample after dissolution was transferred to an analytical vial and analyzed using a triple quadrupole LC / MS / MS system (manufactured by Shimadzu Corporation, LCMS-8080) under the conditions shown below.

<LC conditions>
Analysis column: Shim-pack XR-ODS II (column inner diameter: 2.0 mm × column length: 150 mm, particle diameter: 2.2 μm) manufactured by Shimadzu Corporation
Flow rate: 0.3 mL / min Sample injection volume: 5 μL
Column temperature: 40 ° C
Mobile phase: (A) 5 mM ammonium formate + 0.1% formic acid aqueous solution
(B) 0.1% formic acid acetonitrile solution Gradient program: as shown in Table 1 below

<MS conditions>
Nebulizer gas flow rate: 3 L / min Drying gas flow rate: 12 L / min Curtain gas flow rate: 3 L / min Applied voltage +4.5 kV
Probe temperature: 350 ° C
HSID temperature: 250 ° C
Ionization mode: ESI (+)

[Experimental Example 1] Verification using purified protein solution and creation of calibration curve <Examination of MRM conditions>
(Precursor scan)
Using the purified protein solution of mouse-derived PGRN, a measurement sample was prepared by the above procedure, and detection of trypsin digested fragments was attempted by LC / MS / MS analysis. Among the intermediate sequence fragments and end-added fragments shown in FIGS. 3A and 3B, four types of fragments shown in Table 2 were identified. FIG. 7 shows a chromatogram of the precursor scan, and FIGS. 8A to 8D show mass spectra of the precursor scan. 8A to D, it can be seen that precursor ions derived from fragments 1 to 4 shown in Table 2 are detected, respectively. The reason why other fragments could not be identified is that the peptide in the sample is post-translationally modified (for example, sugar chain modification, etc.), so the m / z predicted from the amino acid sequence and the actual sample It is conceivable that m / z was different.

(Product ion scan)
Four fragments shown in Table 2 were selected as precursor ions, and a product ion scan was performed. MS spectra (MS ² ) are shown in FIGS. 9A to 9D also show product ion information (cleavage position) assigned from the MS spectrum. From these results, it was confirmed that all of the four precursor ions subjected to the product ion scan were fragments derived from PGRN.

(Derivation of MRM conditions and creation of calibration curve)
In each MS spectrum of FIGS. 8A to 8D, three precursor ions assigned to fragments 1 to 4 are selected from those having a high peak intensity in each MS spectrum, and a product ion for each precursor ion. Scanned. Further, among the product ions assigned by each product ion scan, three of the MS ions having the highest peak intensity in each MS spectrum were selected, and the reproducibility of the peak intensity and the linearity of the peak area with respect to the PGRN concentration were confirmed. As a result, for each of the fragments 1 to 4, combinations of precursor ion and product ion mass-to-charge ratio (m / z) shown in Table 3 are selected by selecting a product ion that provides a highly reliable quantitative property. (MRM conditions) was obtained.

<Creation of calibration curve>
MRM analysis was performed under the conditions shown in Table 3 while changing the PGRN concentration. When the peak area of the product ion with respect to the fragment concentration was plotted, as shown in FIGS. 10A to 10D, a linear relationship was found between the PGRN concentration and the peak area, and a calibration curve for each product ion was obtained.

[Experimental example 2] Spike test A purified product of purified mouse PGRN was added to human serum, a measurement sample was prepared by the above procedure, and MRM analysis was performed by LC / MS / MS under the conditions shown in Table 3. The amount of mouse PGRN added to human serum was analyzed as 0 μg / mL (no addition), 0.1 μg / mL, 1 μg / mL, 2 μg / mL. As shown in FIGS. The peak corresponding to indicates an intensity change depending on the amount of PGRN added. In the spike test on human serum containing human PGRN and other various contaminants, the area intensity change depending on the amount of mouse PGRN added was observed. It was confirmed to be effective for the quantification of mouse PGRN and PGRN-derived peptides.

[Example 1] Quantification of PGRN and GRN peptide in mouse serum Using normal mouse serum as a sample, a measurement sample was prepared by the above procedure, and MRM analysis was performed by LC / MS / MS under the conditions shown in Table 3 As shown in FIGS. 12A to 12D, the peak of the tryptic peptide derived from mouse PGRN was confirmed. FIG. 13A shows the result of calculating the peptide concentration from each peak area based on the calibration curves of FIGS. In addition, FIG. 13 (B) shows the GRN3 peptide concentration and GRN4 peptide concentration in mouse serum calculated by the following formulas (1) and (2).
(1) GRN3 concentration = [concentration of fragment 2] − [concentration of fragment 1]
(2) GRN4 concentration = [concentration of fragment 3] − [concentration of fragment 4]

  As shown in the above Examples, it can be seen that the concentrations of GRN3 peptide and GRN4 peptide in mouse serum can be individually quantified by the analysis method of the present invention using the MRM conditions shown in Table 3.

1 LC
2 Ion Source 3 First Stage Quadrupole 4 Second Stage Quadrupole 9 Collision Cell 5 Third Stage Quadrupole 6 Detector 30 Analyzing Room 40 Data Processing Unit 50 Control Unit 51 Operation Unit 52 Display Unit 60 Storage Unit 61 MRM condition table

Claims (8)

The content of the grayed La New phosphopeptide derived from a living organism sample, and an analytical method quantifying using mass spectrometer,
Providing at biological sample containing analyzes all-out peptide;
A step of selectively cleaving a protein and a peptide in the sample at a predetermined amino acid position to obtain a plurality of types of peptide fragments;
The sample after the cutting treatment is
A first mass separation unit for selecting ions having a specific mass-to-charge ratio as precursor ions;
An ion cleavage portion that cleaves each of the selected precursor ions into a plurality of types of product ions;
A second mass separation unit for sorting the product ions based on a mass-to-charge ratio;
A detection unit for detecting and quantifying product ions having a specific mass-to-charge ratio selected by the second mass separation unit;
A step of detecting and quantifying two or more kinds of predetermined product ions; and calculating a content of the predetermined peptide fragment in the sample based on the detected amount of each product ion Steps, and
In the step of detecting and quantifying the predetermined product ion,
(A) Granulin-intermediate sequence comprising at least 6 consecutive amino acids in the amino acid sequence of the granulin peptide and not including any of the N-terminal sequence portion and the C-terminal sequence amino acid sequence portion of the amino acid sequence of the granulin peptide A fragment, and (B1) a granulin-N-terminal addition fragment in which three or more amino acids are further bonded to the N-terminus of at least three amino acid sequences continuous from the N-terminal side of the amino acid sequence of the granulin peptide, and (B2) One selected from the group consisting of a granulin-C-terminal addition fragment in which 3 or more amino acids are further bonded to the C-terminus of at least 3 amino acid sequences continuous from the C-terminal side of the amino acid sequence of the granulin peptide The above end-added fragments;
Product ions generated by cleavage of precursor ions derived from each of two or more peptide fragments, including
In the step of the content of the peptide fragment is calculated, the amount or al of each product ions granulin - calculating an intermediate sequence fragment weight and terminal addition fragment weight, based on the difference between these fragments amount, in the sample An analysis method in which the content of the granulin peptide is calculated. The granulin peptides are granulin 3 peptide and granulin 4 peptide,
In the step of detecting and quantifying the predetermined product ion,
(A1) Granulin 3-intermediate consisting of at least 6 consecutive amino acids in the amino acid sequence of granulin 3 peptide, and including neither the N-terminal sequence portion nor the C-terminal sequence amino acid sequence portion of the amino acid sequence of the granulin 3 peptide Sequence fragments;
(A2) Granulin 4-middle consisting of at least 6 consecutive amino acids in the amino acid sequence of granulin 4 peptide and not including any of the N-terminal sequence portion and the C-terminal sequence amino acid sequence portion of the amino acid sequence of the granulin 4 peptide (B1) a granulin 3-N-terminal addition fragment in which three or more amino acids are further linked to the N-terminus of at least three amino acid sequences continuous from the N-terminal side of the amino acid sequence of the granulin 3 peptide; B2) selected from the group consisting of a granulin 4-C-terminal addition fragment in which 3 or more amino acids are further linked to the C-terminus of at least 3 amino acid sequences continuous from the C-terminal side of the amino acid sequence of the granulin 4 peptide One or more end-added fragments ;
Product ions generated by the cleavage of precursor ions derived from each of the three or more peptide fragments, including
In the step of calculating the content of the peptide fragment,
Wherein the amount or these respective product ions, calculates the granulin 3 intermediate sequence fragment weight and terminal addition fragment weight, based on the difference between these fragments amount, the content of the granulin 3 peptide in the sample is calculated,
The amount or al of each product ion calculates the granulin 4 intermediate sequence fragment weight and terminal addition fragment weight, based on the difference between these fragments amount, the content of the granulin 4 peptides in the sample is calculated The analysis method according to claim 1. In the step of detecting and quantifying the predetermined product ion, it is generated by cleavage of a precursor ion derived from each of the granulin 3-N end addition fragment (B1) and the granulin 4-C end addition fragment (B2). Product ions are quantified,
In the step of calculating the content of the peptide fragment,
The amount or al of each product ion calculates the granulin 3 intermediate sequence fragment mass and granulin 3-N-terminal addition fragment weight, based on the difference between these fragments amount, the content of the granulin 3 peptide in the sample Is calculated,
The amount or al of each product ion calculates the granulin 4 intermediate sequence fragment mass and granulin 4-C-terminal addition fragment weight, based on the difference between these fragments amount, the content of the granulin 4 peptides in the sample The analysis method according to claim 2, wherein is calculated.   The analysis method according to claim 2 or 3, wherein the selective cleavage process at the position of the predetermined amino acid is a process in which peptide bonds on the C-terminal side of lysine residues and arginine residues are selectively cleaved.   The analysis method according to claim 1, wherein the sample is a sample derived from a human or a mouse. The sample is a mouse-derived sample;
The granulin 3-intermediate sequence fragment (A1) has an amino acid sequence represented by SEQ ID NO: 8 in the sequence listing,
The granulin 4-intermediate sequence fragment (A2) has the amino acid sequence represented by SEQ ID NO: 9 in the sequence listing,
The granulin 3-N terminal addition fragment (B1) has the amino acid sequence represented by SEQ ID NO: 7 in the sequence listing,
The analysis method according to any one of claims 2 to 4, wherein the granulin 4-C terminal addition fragment (B2) has an amino acid sequence represented by SEQ ID NO: 10 in the sequence listing.   The analysis method according to any one of claims 1 to 6, wherein the sample is blood, serum, tissue extract, cell extract, urine or cerebrospinal fluid. A first mass separation unit that selects ions having a specific mass-to-charge ratio as precursor ions; an ion-cleavage unit that cleaves each of the selected precursor ions into a plurality of types of product ions; and the product ions based on the mass-to-charge ratio A second mass separation unit for sorting and detecting; a detection unit for detecting and quantifying product ions having a specific mass-to-charge ratio sorted by the second mass separation unit; and the first mass separation unit and the ion cleavage unit the second mass separator unit, and with executing a control to analyze the detection unit, the control unit comprising a computer to analyze the resulting data, using a mass spectrometer comprising, grayed the biological sample A program used for quantitative analysis of the content of lanulin peptide,
In the first mass separation part, any one of (A) the amino acid sequence of the granulin peptide consisting of at least 6 consecutive amino acids and the N-terminal sequence portion and the C-terminal sequence amino acid sequence portion of the amino acid sequence of the granulin peptide And (B1) granulin-N in which three or more amino acids are further bound to the N-terminus of at least three amino acid sequences continuous from the N-terminal side of the amino acid sequence of the granulin peptide. A terminal addition fragment, and (B2) a granulin-C terminal addition fragment in which three or more amino acids are further linked to the C terminus of at least three amino acid sequences continuous from the C terminal side of the amino acid sequence of the granulin peptide. One or more powders selected from the group Additional fragment of the precursor ions from each, the precursor ion selection step of selecting, based on the mass-to-charge ratio which is predetermined for each precursor ion;
In the second mass separation unit, product ions generated by cleavage of the precursor ions selected in the precursor ion selection step are selected based on a mass-to-charge ratio predetermined for each product ion. A product ion detection step of detecting and quantifying each product ion selected in the product ion selection step by the detection unit;
Is executed by the computer, and
Calculating the content of the granulin-intermediate sequence fragment from the amount of product ions produced by the cleavage of the granulin-intermediate sequence fragment quantified in the product ion detection step;
Calculating the content of end-added fragments from the amount of product ions generated by cleavage of the end-added fragments quantified in the product ion detection step; and
Calculating the content of the granulin peptide in the sample from the difference between the content of the granulin-intermediate sequence fragment and the content of the terminal addition fragment;
Is executed by the computer .

Images