JP4775714B2 - Protein identification method and protein identification apparatus - Google Patents

Description

  The present invention relates to a protein identification method and a protein identification apparatus based on peptide fragment analysis.

  To determine the function of a protein, it is necessary to analyze the primary structure of the protein. In order to analyze the primary structure of this protein, it is necessary to cleave the protein and make it into a peptide. However, since there are various proteins, it is difficult to uniformly form a peptide.

  In order to efficiently degrade proteins and recover peptides with good yield, (1) to denature proteins and reduce heterogeneity of degradation due to steric hindrance, and (2) between molecules in the protein molecule after reduction. It is important to protect the cysteine residue SH group, which is rich in reactivity, by cleaving the S—S bond of (3), and (3) to use a proteolytic enzyme with high residue specificity. In chemistry, it is common to prepare a fragmented peptide sample by denaturing and reductively alkylating the protein sample and then digesting with a proteolytic enzyme.

  These denaturing agents and alkylating agents inhibit many proteolytic enzymes, so when operating in solution, dilute to a denaturing agent / alkylating agent concentration acceptable by the enzyme, or use reverse-phase chromatography. It is known that it is necessary to remove denaturing agents and alkylating agents by a graphic method, a dialysis method, an organic solvent precipitation method, and the like. (See Patent Document 1)

As a method using the reverse phase chromatography method, a configuration is proposed in which enzyme-digested peptide fragments are concentrated and desalted with a reverse phase chromatography column, trapped on the column, and then introduced into a reverse phase chromatography-mass spectrometer. ing. (See Non-Patent Document 1)
Japanese Patent Laying-Open No. 2004-301715 (paragraphs 0005 and 0006) LC PACKINGS Application Note UltiMate Capillary and Nano LC system, Proteomics # 8 “AUTOMATED 2D LC COUPLED TO ESI-MS / MS FOR THE ANALYSIS OF COMPLEX PEPTIED SAMPLES

  In protein fragmentation by peptideization, fragmentation may be incomplete due to insufficient enzymatic digestion. Such incomplete fragmentation results in peptide fragments having a large molecular weight such as uncut protein.

  This peptide fragment having a large molecular weight is adsorbed on a reverse phase chromatography column for peptide separation and adversely affects the separation, and also causes a deterioration of the column itself.

  In addition, the presence of peptide fragments with large molecular weight caused by incomplete fragmentation may cause measurement errors such as complicated mass spectra and double acquisition of the same information. There is a problem of hindering.

  In particular, in the column switching system in which the introduced protein is subjected to reductive alkylation online, followed by enzymatic digestion and peptide separation-mass spectrometry, the enzymatic digestion is often insufficient, and the above-described problems become significant.

  The problem of generation of peptide fragments having a large molecular weight due to insufficient peptide fragmentation is not limited to peptide fragmentation by enzyme digestion, but also occurs in chemical fragmentation by chemical techniques.

  In view of the above, an object of the present invention is to solve the above problems, eliminate peptide fragments having a large molecular weight that hinder analysis, and perform protein identification using peptide fragments having a molecular weight suitable for analysis.

  In the present invention, the peptide mixture is concentrated on the RAM column in the stage before the reverse phase chromatography separation, so that it is not adsorbed on the reverse phase chromatography column for peptide separation and the sequence information for identifying the protein is not complicated or increased. Only a relatively small molecular weight peptide fragment is trapped on a RAM column, while the protein residue and the sequence information are complicated by adsorbing to a reversed-phase chromatography column for peptide separation and adversely affecting peptide separation. The peptide fragment having a large molecular weight that is increased is not trapped by passing through this RAM column, thereby achieving the object of identifying the protein by the peptide fragment having a molecular weight suitable for analysis.

  This invention can be made into the method aspect of a protein identification method, and the apparatus aspect of a protein identification apparatus.

  The embodiment of the protein identification method of the present invention is to identify a protein by peptide fragmentation of the target protein, separation of the peptide fragment from the peptide mixture obtained by this peptide fragmentation by reverse phase chromatography, and mass spectrometry In the method, a step of introducing a peptide mixture after peptide fragmentation into a concentration RAM column, a step of trapping a peptide fragment having a low molecular weight in a concentration RAM column, a peptide fragment having a large molecular weight and the remaining protein in a concentration RAM column A step of passing through and discarding, a step of introducing the peptide fragment trapped in the RAM column for concentration into a reverse phase chromatography column for peptide separation and reverse phase separation, and a step of introducing the separated peptide fragment into mass spectrometry.

  The concentration RAM column allows peptide fragments with a large molecular weight and residual proteins to pass through, traps only peptide fragments with a low molecular weight, and introduces the trapped peptide fragments into a reversed-phase chromatography column for peptide separation. Protein fragments that can adversely affect the separation of proteins can be prevented from being introduced into reverse-phase chromatography separation columns, and peptide fragments with large molecular weights that can complicate and increase sequence information are introduced into mass spectrometry. Can be prevented.

  The peptide fragment can be obtained by chemical peptide fragmentation as well as by enzymatic digestion peptide fragmentation. In enzymatic digestion peptide fragmentation, the target protein is reductively alkylated and then digested with a proteolytic enzyme.

  In addition, the protein identification apparatus of the present invention is a protein identification that identifies a protein by peptide fragmentation of the target protein, separation of the peptide fragment from the peptide mixture obtained by this peptide fragmentation by reverse phase chromatography, and mass spectrometry A concentration RAM column for introducing a peptide mixture after peptide fragmentation and trapping the peptide fragments, and a reverse phase chromatography for peptide separation for separating the peptides by introducing the peptide fragments trapped in the concentration RAM column. Introducing a peptide mixture into a chromatography column, a RAM column for concentration, a switching valve for deriving peptide fragments trapped from the RAM column for concentration, and a peptide separated by a reversed-phase chromatography column for peptide separation, mass spectrometry A mass spectrometer for performing .

  Here, the switching valve has an introduction port for introducing the peptide mixture, a discharge port, two connection ports connected to the RAM column for concentration, and a connection port connected to the reverse phase chromatography column for peptide separation. This switching valve introduces the peptide mixture into the concentration RAM column by switching the connection port between the introduction port and the discharge port to the connection port of the concentration RAM column, traps a peptide fragment having a small molecular weight, and By switching the connection port to connect the connection port connected to the peptide separation reverse phase chromatography column, the peptide fragment trapped in the concentration RAM column is led to the peptide separation reverse phase chromatography column.

  In the concentration RAM column of the present invention, a peptide fragment having a small molecular weight is trapped on the concentration RAM column, and a peptide fragment having a large molecular weight and the remaining protein are passed through the concentration RAM column. This prevents large amounts of peptide fragments from being introduced into the mass spectrometry, which prevents the introduction of protein residues that adversely affect peptide separation into the reversed-phase chromatography separation column and complicates and increases the sequence information. To prevent it. This concentration RAM column can be configured to be filled with a contact limiting filler.

  In column switching systems that perform reductive alkylation of introduced proteins online, then perform enzyme digestion, and perform peptide separation-mass spectrometry, enzyme digestion is often insufficient. It becomes more prominent.

  According to the present invention, peptide fragments having large molecular weights that hinder analysis can be excluded, and protein identification can be performed using peptide fragments having molecular weights suitable for analysis.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a diagram for explaining a schematic configuration of a protein identification apparatus of the present invention.

  In FIG. 1, a protein identification apparatus 1 of the present invention introduces a concentration RAM column 2 for trapping peptide fragments by introducing a peptide mixture after peptide fragmentation, and a peptide fragment trapped in the concentration RAM column 2. An ionization unit 3 that separates and ionizes the peptide, a mass spectrometer 5 that introduces the peptide separated by the ionization unit 3 and performs mass spectrometry, an introduction of the peptide mixture into the RAM column 2 for concentration, and a RAM for concentration A switching valve 4 for switching the peptide fragment trapped in the column 2 to the ionization unit 3 is provided.

  The switching valve 4 includes an introduction port 4a for introducing the peptide mixture, an exhaust port 4b, two connection ports 4c and 4f for connecting the concentration RAM column 2, and a connection port 4e for connecting the ionization unit 3. A port 4d for introducing a mobile phase is provided.

  An injector 14 is connected to the introduction port 4a. A sample to be identified is injected into the injector 14 into the sample introduction liquid 21 sent by the liquid feed pump 11 and introduced into the introduction port 4 a of the switching valve 4. As a sample to be injected into the injector 14, for example, a fragmented peptide obtained by reductive alkylation of a target protein and then digested by a proteolytic enzyme or fragmented by chemical fragmentation is used.

  The mobile phases 22 and 23 are introduced into the port 4d for introducing the mobile phase by the liquid feed pumps 12 and 13, respectively. A joint 16 to which a gradient mixer 15 and a resistance tube 17 are connected is connected between the liquid feed pumps 12 and 13 and the port 4d so that the gradient by the mobile phases 22 and 23 can be performed.

  A degasser 18 may be provided between the sample introduction liquid 21 and the mobile phase liquids 22 and 23 and the liquid feed pumps 11, 12, and 13. By providing the degasser 18, oxygen molecules and nitrogen molecules contained in the sample introduction liquid 21 and the mobile phases 22 and 23 are discharged, and generation of bubbles in the downstream channel can be prevented.

  The concentration RAM column 2 is connected to both ends of the connection ports 4c and 4f. In addition, the discharge port 4b is connected to one end of the concentration RAM column 2 by switching the switching valve 4, and removes a large molecular weight peptide fragment or protein residue that has passed through the concentration RAM column 2 together with the sample introduction solution 21. Discharge.

  In addition, the outlet port 4f is connected to the ionization unit 3 through the filter 19 and is connected to the concentration RAM column 2 by switching the switching valve 4, and is trapped in the concentration RAM column 2 together with the mobile phases 22 and 23. The obtained peptide fragment is introduced into the ionization part 3.

  The ionization unit 3 is an electrospray capillary (hereinafter referred to as a peptide separation reverse phase chromatography column 3b and a peptide separation reverse phase chromatography column) that combines the peptide fragment multiprotonated molecules introduced from the concentration RAM column 2 with a peptide separation reverse phase chromatography column. And then ionized by the electrospray unit 3a and introduced into the mass spectrometer 5.

  Here, the concentration RAM column 2 is a column having a property of allowing a high molecular weight compound with a high molecular weight to pass through without being held and holding only a low molecular weight compound with a low molecular weight, and has a special surface structure and pore size. Thus, a protein that does not enter the pores can be passed while retaining the target component having a low molecular weight under certain conditions. In the present invention, by applying this concentration RAM column to a peptide mapping system using enzyme-digested peptide fragments, it is possible to selectively remove uncut proteins and peptide fragments having a large molecular weight caused by incomplete fragmentation. it can.

  Examples of this column include Shim-pack MAYI-ODS (Shimadzu Corporation), ISPR-GFF (Regis Technologies Inc), and Lichrospher PR-18 ADS (Merck). Shim-pack MAYI-ODS is a surface structure in which the outer surface of silica gel is coated with a hydrophilic polymer and only the inside of the pores is chemically decorated with octadecyl groups (ODS). In addition, because it is blocked by the hydrophilic polymer on the outer surface, it elutes without being retained in the stationary phase ODS, while the induced low molecular weight compound penetrates into the pores and is retained in the stationary phase on the inner surface. It is what is done.

  Next, an operation example of the protein identification device of the present invention will be described with reference to FIGS.

  First, the flow path of the injector 14 is connected to the introduction port 4a by switching the switching valve 4 (A in FIG. 2). The target protein is peptide fragmented, a peptide mixture obtained by this peptide fragmentation is prepared, this peptide mixture is injected from the injector 14 and introduced into the introduction port 4a together with the sample introduction solution 21.

  Peptide fragmentation can use, for example, enzymatic digestion peptide fragmentation in which the target protein is reductively alkylated and then digested with a proteolytic enzyme, or chemical fragmentation by a chemical technique (B in FIG. 2).

  The peptide mixture (protein enzyme digest) introduced from the introduction port 4a is introduced into the concentration RAM column 2 from the connection port f and discharged through the connection port 4c and the discharge port 4b (D in FIG. 2). By continuing to feed the sample introduction liquid 21 by driving the liquid feed pump 11, the concentration RAM column 2 performs desalting, removal of uncut protein, peptide having a large molecular weight on the introduced peptide mixture. The fragments are removed and the low molecular weight peptide fragments are concentrated and retained (C in FIG. 2).

  Next, the switching valve 4 is switched, and the flow path of the gradient mixer 15 is connected to the introduction port 4d (E in FIG. 3). The liquid feeding pumps 12 and 13 are driven to start the liquid feeding of the mobile phases 22 and 23, and the mobile phase is introduced into the concentration RAM column 2 through the connection ports 4d and 4c (F in FIG. 3). The peptide fragment trapped in the column 2 is introduced into the ionization section 3 through the connection port 4f and the outlet port 4e together with the mobile phase (G in FIG. 3).

  The ionization unit 3 separates and ionizes the introduced peptide fragment and introduces it into the mass spectrometer 5 (H in FIG. 3).

  In the example shown in FIGS. 1 to 3, in the configuration in which peptide fragments are separated by reverse phase chromatography and then detected by a mass spectrometer, the mass spectrometer is ionized by an electrospray method. Not only the spray method but also separation by reverse phase chromatography, fractionation with a fraction collector, and ionization by MALDI method (Matrix Assessed Laser Desortion / Ionization) may be used.

  FIG. 4 is a diagram for explaining another example of the schematic configuration of the protein identification device of the present invention. 4, the configuration up to the filter 19 is the same as the configuration shown in FIG. 1, so the configuration of the filter 19 will be described. The configuration shown in FIG. 1 is an online configuration, whereas the configuration shown in FIG. 4 is an offline configuration.

  The peptide fragment trapped in the RAM column 4 is introduced into the analysis column 31 through the connection port 4f and the outlet port 4e together with the mobile phase. The analytical column 31 separates the introduced peptide fragment and passes a detector 32 such as UV. Sort by peak or time detected by detector 32. Introduce into fraction collector or MALDI plate spotter 33.

  Peptide components or fractions fractionated by the fraction collector or MALDI plate spotter 33 are introduced into a MALDI (Matrix Assessed Laser Desortion / Ionization) -TOFMS (Time of Flight Mass Spectrometry) device 34. . Examples of the fraction collector include Shimadzu FRC-10A (trade name), and examples of the MALDI plate spotter include Shimadzu AccuSpot (trade name).

It is a figure for demonstrating schematic structure of the protein identification apparatus of this invention. It is a figure for demonstrating the operation example of the protein identification apparatus of this invention. It is a figure for demonstrating the operation example of the protein identification apparatus of this invention. It is a figure for demonstrating another example of schematic structure of the protein identification apparatus of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Protein identification apparatus, 2 ... Condensation RAM column, 3 ... Ionization part, 3a ... Electrospray unit for peptide fragments, 3b ... Reverse phase chromatography column for peptide separation, 4 ... Switching valve 4, 4a ... Introduction port, 4b ... discharge port, 4c ... connection port, 4d ... mobile phase introduction port, 4e ... derivation port, 4f ... connection port, 5 ... mass spectrometer, 11, 12, 13 ... liquid feed pump, 14 ... injector, 15 ... gradient mixer , 16 ... T joint, 17 ... Resistance tube, 18 ... Dessa, 21 ... Sample introduction solution, 22, 23 ... Mobile phase, 31 ... Analysis column, 32 ... Detector, 33 ... Fraction collector, MALDI plate spotter, 34 ... MALDI-TOFMS device.

Claims (5)

In a protein identification method for identifying a protein by subjecting the target protein to peptide fragmentation, separating the peptide fragment from the peptide mixture obtained by this peptide fragmentation by reverse phase chromatography, and mass spectrometry,
Introducing the peptide mixture after peptide fragmentation into a RAM column for concentration,
The peptide fragment with a low molecular weight is trapped on a concentration RAM column, the peptide fragment with a high molecular weight and the remaining protein are passed through the concentration RAM column and discarded.
A protein identification method comprising introducing a peptide fragment trapped in a concentration RAM column into a reversed-phase chromatography column for peptide separation, reverse-phase separation, and introducing the separated peptide fragment into mass spectrometry.   The protein identification method according to claim 1, wherein the peptide fragmentation is enzyme digestion peptide fragmentation in which a target protein is reductively alkylated and then digested with a proteolytic enzyme.   The protein identification method according to claim 1, wherein the peptide fragmentation is chemical fragmentation. In a protein identification apparatus for identifying a protein by subjecting the target protein to peptide fragmentation, separating the peptide fragment from the peptide mixture obtained by this peptide fragmentation by reverse phase chromatography, and mass spectrometry,
A concentration RAM column for introducing a peptide mixture after peptide fragmentation to trap peptide fragments;
A peptide separation reverse phase chromatography column for separating peptides by introducing the trapped peptide fragments into the concentration RAM column;
A RAM column for concentrating peptide mixtures, having an introduction port for introducing a peptide mixture, an exhaust port, two connection ports for connecting to a RAM column for concentration, and a connection port for connecting to a reverse phase chromatography column for peptide separation A switching valve that switches between the introduction of the peptide fragment and the peptide fragment trapped in the concentration RAM column to the reverse phase chromatography column for peptide separation,
A mass spectrometer for performing mass spectrometry by introducing the peptide separated by the reverse phase chromatography column for peptide separation,
The concentration RAM column is
Trap peptide fragments with low molecular weight on a RAM column for concentration,
A protein identification apparatus characterized in that a peptide fragment having a large molecular weight and the remaining protein are passed through a concentration RAM column.   The protein identification apparatus according to claim 4, wherein the concentration RAM column is filled with a contact limiting filler.

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