WO2007108140A1 - Procede d'identification et de quantification pour un echantillon de proteine a l'aide d'une electrophorese et d'une spectrometrie de masse - Google Patents

Procede d'identification et de quantification pour un echantillon de proteine a l'aide d'une electrophorese et d'une spectrometrie de masse Download PDF

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WO2007108140A1
WO2007108140A1 PCT/JP2006/306396 JP2006306396W WO2007108140A1 WO 2007108140 A1 WO2007108140 A1 WO 2007108140A1 JP 2006306396 W JP2006306396 W JP 2006306396W WO 2007108140 A1 WO2007108140 A1 WO 2007108140A1
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sample
protein
group
modifying
carrier
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PCT/JP2006/306396
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English (en)
Japanese (ja)
Inventor
Kumi Matsumoto
Iwao Ohtsu
Hiroyuki Fukuda
Shinichiro Kobayashi
Hiroki Kuyama
Chikako Toda
Eiichi Matsuo
Toshikazu Minohata
Osamu Nishimura
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Shimadzu Corporation
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Priority to PCT/JP2006/306396 priority Critical patent/WO2007108140A1/fr
Publication of WO2007108140A1 publication Critical patent/WO2007108140A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44739—Collecting the separated zones, e.g. blotting to a membrane or punching of gel spots

Definitions

  • the present invention relates to proteomics, that is, a comprehensive analysis method of proteins. More specifically, the present invention relates to a method for identification and quantification of protein using electrophoresis and mass spectrometry using electrophoresis and mass spectrometry.
  • 2005-189104 describes a method of concentrating and separating a protein peptide modified with an NBS reagent using a carrier having a phenyl group.
  • a peptide modified with an NBS reagent is designated as ⁇ -cyan-3-hydroxygay cinnamate, 3-hydroxy-4-hydroxybenzoate, or 3-hydroxy-1 4 —A mixture of nitrobenzoic acid and ⁇ -cyano 4-hydroxygaynamic acid is used as a matrix and measured using a MALD I mass spectrometer to efficiently detect peptides modified with NBS reagent .
  • NBS-labeled peptides that is, NBS group-containing peptides
  • NBS reagent modifies tributophane residues that are abundant in proteins
  • peptides in MS samples will be labeled with NBS-unlabeled peptides (ie, NBS Group-free peptides).
  • NBS Group-free peptides A large amount of NBS group-free peptide causes ion subsidence for the NBS group-containing peptide to be detected by mass spectrometry. For this reason, the detection efficiency of a NB S group containing peptide worsens.
  • an object of the present invention is to provide a proteome analysis method using an electrophoresis method, which enables differential display with a single gel. is there. Furthermore, a further object of the present invention is to provide a proteome analysis method using an electrophoresis method, which provides a quantitative reliability and sensitivity.
  • the present inventor has found that the object of the present invention can be achieved by removing the NBS group-free peptide and concentrating the NBS group-containing peptide. Furthermore, the present inventor has found that the object of the present invention and the further object can be achieved by using a matrix more suitable for detection of NBS group-containing peptides by mass spectrometry. Based on these findings, the present invention has been completed.
  • the present invention includes the following inventions.
  • the following (1) to (5) are directed to methods for identifying and quantifying protein samples.
  • the following (6) is directed to a separation device that can be usefully used in the methods (1) to (5).
  • the following (7) is directed to a kit that can be usefully used in the methods (1) to (5).
  • a desired protein spot is cut out from the plurality of protein spots, the cut out protein spot is fragmented in the gel, and the peptide fragment containing the modifying group and other peptide fragments are concentrated. Obtaining a mixed sample for separation;
  • a method for identifying and quantifying protein samples comprising:
  • the term “protein” is used to include a peptide having a relatively small molecular weight.
  • step (i) two types of protein samples are prepared in step (i), and the two types of protein samples are separately prepared using two types of modifying reagents that differ only in the isotopic composition in step (ii).
  • Modified ie, isotope-labeled one of the protein samples by the isotope labeling method
  • step (iii) electrophoresed in step (iV)
  • step (vii) fragmentation in the gel was performed, and in step (vi) the modified peptide Is concentrated and separated, and mass spectrometry is performed in step (vii).
  • step (ii) a compound having an aryl group is used as a modifying reagent, and the modified protein into which the aryl group is introduced as a modifying group and the modified protein 1 are obtained.
  • step (vi) The method for identifying and quantifying a protein sample according to (1), wherein a carrier having an aryl group is used as a carrier having a structure capable of interacting with the modifying group.
  • the compound having an aryl group as the modifying reagent is 2-nitro [ 13 C 6 ] benzenesulfuryl chloride and 2-nitro [ 12 C 6 ] benzenesulfuryl chloride.
  • a tip for concentration and separation in which a pipette tip is filled or fixed with a carrier having a phenyl group.
  • a proteome analysis method using electrophoresis which enables a differential display with a single gel. Furthermore, according to the present invention, there is provided a proteome analysis method using an electrophoresis method, and an analysis method with high reliability and sensitivity of quantification.
  • Figure 1 shows the results obtained by subjecting sample 1 obtained by electrophoresis and in-gel digestion to a protein sample labeled with an isotope labeling method using NBS reagent, and desalting purification using Ziptip. This is the MA LD I—TOF MS spectrum of the sample for mass spectrometry.
  • Figure 2 shows a sample sample obtained by electrophoresis and in-gel digestion of a protein sample labeled by the isotope labeling method using NBS reagent.
  • 2 is a MA LD I-TOF MS spectrum of a sample for mass spectrometry obtained by concentrating and separating an NBS group-containing peptide fragment by ⁇ .
  • Figure 3 shows protein samples labeled with the isotope labeling method using NBS reagent.
  • the sample is obtained using the MA LD I-TOF MS spectrum of the sample for mass spectrometry obtained by desalting and purification using Ziptip on sample 2 obtained by electrophoresis and in-gel digestion.
  • Figure 4 shows sample 2 obtained by electrophoresis and in-gel digestion of a protein sample labeled with an isotope labeling method using an NBS reagent. Desalting and purification using Ziptip and NBS group using Phenyltip 2 is a MALD I-TOF MS spectrum of a sample for mass spectrometry obtained by concentrating and separating a peptide fragment contained therein.
  • Figure 5 shows Pheny's analysis of NBS group-containing peptide fragments by ip against sample 2 obtained by electrophoresis and in-gel digestion of a protein sample labeled by the isotope labeling method using NBS reagent. It is a MA LD I-TOF MSZMS spectrum of the sample for mass spectrometry obtained by performing concentration separation.
  • Figure 6 is obtained by desalting and purifying Sample 3 obtained by electrophoresis and in-gel digestion of a protein sample labeled with an isotope labeling method using NBS reagent. This is a MA LD I-TO F MS spectrum of a sample for mass spectrometry.
  • Figure 7 shows sample 3 obtained by electrophoresis and in-gel digestion of a protein sample labeled by the isotope labeling method using NBS reagent, and desalted by Ziptip and NBS group by Phenyltip. It is a MALD I-TOF MS spectrum of a sample for mass spectrometry obtained by concentration separation of peptide fragments.
  • the present invention provides protein identification and quantification methods. Identification and quantification method of the present invention
  • the step (i) of preparing two kinds of protein samples two kinds of proteins are modified separately using a modification reagent that differs only in the isotopic composition (that is, one of the two isotopes is labeled by the isotope labeling method).
  • Labeling a protein sample) step (ii) mixing two modified protein samples (iii), performing electrophoresis (iv), performing gel fragmentation, and step (V)
  • one protein is a protein sample from one sample and the other protein is a protein sample from another sample; one protein is the protein sample to be analyzed and the other protein sample is When it is a control protein for said one protein; when one protein sample is a protein sample extracted from a pathologic sample and the other protein sample is a protein sample extracted from a normal sample, etc. .
  • the protein sample may be collected and / or extracted from an individual and then solubilized.
  • the solubilization method is not particularly limited.
  • a protein can be solubilized using a surfactant such as sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride, etc. as a denaturing agent.
  • SDS sodium dodecyl sulfate
  • urea urea
  • guanidine hydrochloride etc.
  • concentration of denaturing agent is not particularly limited, so that protein samples can be solubilized and denatured.
  • a person skilled in the art may determine as appropriate in consideration of the type of sample and other conditions.
  • the reaction conditions may be appropriately determined by those skilled in the art in consideration of the modifying agent to be used regardless of normal temperature modification or heat modification.
  • one of the two proteins isotope-labeled by the so-called isotope labeling method.
  • an appropriate group is introduced as a modifying group into both of the two types of proteins so that the molecule to be detected in the present invention can be efficiently concentrated and separated in the subsequent step (vi).
  • a specific structure in the protein is used as the modification target.
  • the modifying reagent is particularly limited to a compound having a group capable of selectively modifying the specific structure and capable of interacting with the carrier used in the subsequent step (vi). It can be used without using it.
  • a triftophan residue in a protein may be used as a modified target.
  • the modifying reagent in the present invention uses a combination of two compounds having the same molecular structure but having different molecular weights by containing isotopes having different mass numbers.
  • compounds with higher molecular weights are used as heavy reagents, and reagents with lower molecular weights are used as light reagents.
  • a compound in which at least one element constituting the modifying reagent molecule is labeled with a stable isotope has the same structure, but the aforementioned element is a stable isotope. Use in combination with a compound labeled with another stable isotope.
  • the compound labeled with a stable isotope having a large mass number is used as a heavier reagent, and the other compound is used as a light reagent.
  • R represents an organic group
  • X represents a leaving group
  • R 2 -X 2 R 2 represents a substituted aralkyl group.
  • X 2 represents a leaving group
  • R 1 S or 1 R 2 is introduced as a modifying group into the protein.
  • the compound represented by the above general formula 1 that is, a sulfinyl compound, aryl sulfenyl halide in which in the above general formula 1 is substituted is a aryl group.
  • arylsulfenyl halide 2-nitrobenzenesulfenyl chloride (NBSG NBS reagent) is preferable.
  • NBSGI heavy 2-nitro [ 13 C 6 ] benzenesulfuryl chloride
  • Toro [ 12 C 6 ] benzenesulfuryl chloride (NBSGI light) is preferable to use in combination with Toro [ 12 C 6 ] benzenesulfuryl chloride (NBSGI light).
  • NBSCI heavy reagent and NBSCI light reagents both manufactured by Shimadzu Corporation 13 G NBS (R) Stable Isotope Label ing Kit - sold housed in N.
  • sulf-L nyl compounds as modifying reagents for tributophan residues in proteins and peptides are described in detail in International Publication No. 2004/002950 / Nflet.
  • benzyl halide is preferable.
  • examples of the benzyl halide include 2-hydroxy-5-nitrobenzyl bromide (2-hydroxy-5-nitrobenzyl bromide).
  • 2-hydroxy-5-nitrobenzylbutamide 2-hydroxy-5-nitro [ 13 C S ] benzyl bromide as a heavy reagent and 2-hydroxy-5- Nitro [ 12 C 6 ] benzylbutamide is preferably used in combination.
  • Heavy reagent light reagent As for the benzyl halide compound as a modification reagent for the tributophane residue in the protein 'peptide, see Horton, HR and Koshland, D.E, Jr. (1972), Modification of proteins with actibenzylyl amide, Methods in It is described in detail in ENZYM0L0GY, 25, 468-482, etc.
  • the modifying reagent is not limited to the compounds listed above, and a person skilled in the art appropriately selects a compound having the ability to modify a specific structure in the protein and is particularly limited as long as it is combined with a heavy reagent and a light reagent. Not.
  • the two modified protein samples I ′ and II ′ obtained by the above modification process are mixed together.
  • the mixed modified protein sample may be appropriately subjected to a treatment such as a reductive alkylation treatment.
  • the reductive alkylation treatment can be performed by a usual method.
  • this process may be performed in the following step (V) instead of being performed in this step.
  • the mixed sample for electrophoresis obtained in this step may use a normal buffer known to those skilled in the art, including Tris-HG, SDS, -mercaptoethanol, glycerol, and the like.
  • the electrophoresis sample obtained by the above process is subjected to electrophoresis.
  • electrophoretic movement a method known to those skilled in the art may be used.
  • isoelectric focusing SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or
  • gel electrophoresis such as two-dimensional electrophoresis (2D-PAGE) combining them, it is developed into multiple protein spots.
  • the detection of the protein after the electrophoresis is preferably performed by Coomassie prioriant blue staining and fluorescence detection.
  • a desired protein spot is cut out from the plurality of protein spots obtained in the above process.
  • In-gel fragmentation is performed on the excised protein spots.
  • the fragmentation method may be enzymatic fragmentation or chemical fragmentation.
  • digestion using an enzyme such as trypsin is performed.
  • the desired protein cleaved is fragmented into a peptide fragment containing a modifying group and other peptide fragments.
  • an N B S reagent is used as a modifying reagent in the above-described step (U)
  • this step results in fragmentation into a peptide fragment having an N B S group and a peptide fragment having no N B S group.
  • the mixture of the thus obtained modifying group-containing peptide fragment and its ⁇ (ya's peptide fragment) is subjected to an appropriate treatment if necessary, and becomes a mixed sample for concentration and separation described later.
  • the treatment to be performed include reduction, alkylation, and desalting treatment, etc.
  • This desalting treatment can be performed using a desalting instrument such as Ziptip (manufactured by Millipore).
  • the modified group-containing peptide fragment is concentrated and separated from the obtained mixed sample for concentration and separation (mixture of modified group-containing peptide fragments and other peptide fragments).
  • concentration and separation step in the present invention is preferable because it enables highly efficient detection of a desired modified group-containing peptide fragment.
  • peptide fragments containing tributophane with a low content in the protein are efficiently concentrated and separated, so that the peptide fragments containing tributophane are detected with higher sensitivity by mass spectrometry than when they are not concentrated and separated. The For this reason, protein quantification is also improved.
  • the concentration / separation method it is preferable to use a separation instrument in which a small instrument such as a pipette tip is filled or fixed with a carrier.
  • a separation device in which a carrier is packed or fixed in a small device such as a pipette tip is very suitable for concentration separation on such a small sample scale.
  • a carrier used in the concentration separation a carrier having a group capable of interacting with the modifying group introduced in the modifying step (U) is used.
  • the interaction here is an action based on affinity such as van der Waals interaction, 7 ⁇ -7 ⁇ electron interaction, hydrophilic interaction and hydrophobic interaction.
  • the carrier having a group capable of interacting with the modifying group ODS, silica gel, cephadex, and the like may be used.
  • ODS organic compound
  • silica gel, cephadex, and the like may be used as the carrier having a group capable of interacting with the modifying group.
  • an ODS-based carrier is used from the viewpoint of workability in the production of the separation instrument.
  • a monolith type carrier with high decomposition performance as the ODS-based carrier.
  • the monolith type ODS column differs from the widely used packed column in that the framework on the 3D network and its voids (ie, the flow path) are integrated.
  • the carrier and the column are in the form of a body and do not require frit.
  • arylsulfenyl which is a preferred form of the compound represented by the above-described modification step (wherein i represents general formula 1: F ⁇ —S—X, represents an organic group and X represents a leaving group)
  • an R, 1 S— group (in this case is an aryl group) is introduced as a modifying group.
  • R 2 _X 2 (R 2 represents an aralkyl group, X 2 represents a leaving group), the R 2 — group is introduced as a modifying group
  • a ⁇ -electron group such as a modifying group having an aryl group.
  • a carrier having an aryl group is preferably used as the carrier, and a carrier having a phenyl group is preferable as the carrier having an aryl group, in which case the peptide fragment to be concentrated and separated from the carrier is used.
  • Phenyltip a separation device in which a carrier having a phenyl group is filled in a pipette tip
  • a Phenyltip Further details of Phenyltip will be described in “Concentration separation chip and kit containing it” described later.
  • a combination using an NBS reagent as a modifying reagent in the step (ii) and using a carrier having a phenyl group in the step (vi) is particularly preferable.
  • acetonitrile and 0.1% TFA aqueous solution may be used.
  • chemical species other than the modifying group-containing peptide can be positively removed.
  • the adsorbed modification group-containing peptide fragment is eluted with an appropriate solution and then subjected to an appropriate treatment as necessary to become a sample for the next mass spectrometry.
  • a matrix may be included in the sample for mass spectrometry. The matrix will be described in detail in step (vii) below.
  • the elution solution can be appropriately determined by those skilled in the art depending on the type of the modified modifying group-containing peptide fragment.
  • a hydrophobic group such as an NBS group
  • MALDI mass spectrometry is performed in the step (vii) described later
  • it may be eluted using a matrix solution described later.
  • the matrix (described later) preferably used in the present invention is very compatible with an NBS group-containing peptide, particularly when an NBS group is introduced as a modifying group. Therefore, elution with a preferred matrix solution improves workability for subsequent mass spectrometry measurements.
  • a desired peptide fragment can be eluted more efficiently. Note that, for example, a fractionation process is performed as an appropriate process performed as necessary.
  • the method for desalting can be carried out by methods known to those skilled in the art.
  • Examples of the method for performing fractionation include a method using a system using a reverse phase column and HPLC.
  • the sample for mass spectrometry obtained in the above step is subjected to mass spectrometry.
  • a MALDI mass spectrometer can be used for the measurement in this step.
  • a MALDI-T0F type mass spectrometer eg Shimadzu Corporation / Craitos AXIMA- GFR plus
  • a MAID ⁇ IT-T0F type mass spectrometer eg Shimadzu Corporation / Crates AXIMA-QIT
  • the matrix used for the sample for mass spectrometry is not particularly limited.
  • DHB (2,5-dihydroxybenzoic acid; 2, 5-dihydroxybenzoic acid)
  • 4-CHCA Hydrophilic 4-hydroxycinnamic acid; -cyano-4-hydroxycinnamic acid
  • 3-CHCA —cyano 3-hydroxy Cinnamic acid (a-cyano-3-hydroxycinnamic acid), 3H4NBA (3-hydroxy-4-nitrobenzoic acid), etc.
  • 4-GHGA should be used as the matrix.
  • a mixture of 3H4MBA and / or 3-GHGA and 4-GHGA may be used as a matrix.
  • 3-CHCA and 3H4MBA are preferably used when a hydrophobic ⁇ -electron group such as an NBS group is introduced into the protein in the step (i ⁇ ).
  • a mixed matrix obtained by mixing 3-GHGA or 3 ⁇ 4 ⁇ with 4-CHGA is particularly preferably used when a hydrophobic group such as an NBS group is introduced into a protein in the step (ii).
  • a hydrophobic group such as an NBS group
  • Such a mixed matrix is an excellent matrix having the above-mentioned specific ionization ability and self-decay suppression ability as well as high-sensitivity measurement ability. For this reason, the quantitative reliability of the method of the present invention is very high.
  • the compound used as a matrix can be appropriately determined by those skilled in the art for the purpose of being used as a matrix for mass spectrometry. For example, these compounds can be used as a solution having a concentration of 1 mg / mI to saturation.
  • aqueous solution of acetonitrile As a solvent used for the preparation of such a solution, it is preferable to use an aqueous solution of acetonitrile, an aqueous solution of trifluoroacetic acid (TFA), or an aqueous solution of acetonitrile-trifluoroacetic acid (TFA).
  • the concentration of the acetonitrile is not particularly limited, but 90% or less, preferably about 7 O v / v% can be used.
  • the concentration of TFA is not particularly limited, but it may be 1 ⁇ / ⁇ / 0 or less, preferably about 0.1 ⁇ / ⁇ %.
  • 1 mg / ml to saturated concentration for 4-CHGA preferably 5 to 8 mg / m I; 1 mg / m I to saturated for 3-CHGA concentration
  • the matrix solution is preferably used in a volume ratio of 1:10 to 10 : 1, more preferably 1 : 3 to 3: 1, for example, 1: 1.
  • such a matrix solution can be used for elution of the peptide fragment containing the modified group separated in the step (vi).
  • the use of such an elution solution means that the modifying group-containing peptide fragment to be eluted in the step (vi) 7 ⁇ - ⁇ , which is thought to work with the matrix contained in the solution, has the advantage of efficient elution of the peptide fragment containing the modifying group due to the affinity resulting from electron interaction and hydrophobic interaction. is there.
  • the modification group-containing peptide is detected as a pair peak by MS measurement, and the protein can be quantified from the intensity ratio of two peaks constituting the pair peak.
  • protein identification may be performed by PMF analysis or MSZMS analysis.
  • identification may be performed by PMF analysis and quantification by MS analysis.
  • the sample after gel fragmentation is desalted using Ziptip to prepare a sample for mass spectrometry for identification by PMF analysis.
  • Samples for mass spectrometry can be prepared for quantification by MS analysis by concentrating and separating the modifying group-containing peptide fragments using Phenyltip or the like.
  • PMF solution For mass spectrometry samples for analysis, 4-CHCA is preferably used as the matrix.
  • the sample for mass spectrometry for MS analysis it is preferable to use a mixture of 3 H4 NBA and / or 3-0! To 10 and 4-01 "10 8 as a matrix.
  • Samples for mass spectrometry can be dropped on a single MS plate and used for mass spectrometry measurement, ie, this method requires the preparation of two types of mass spectrometry samples. Identification and quantification can be performed with this mass spectrometer.
  • identification and quantification are performed using two mass spectrometers
  • identification can be performed by MS / MS analysis and quantification can be performed by MS analysis.
  • 'Pheny can concentrate and separate the modifying group-containing peptide fragment using ip or the like on the sample after gel fragmentation to prepare a sample for mass spectrometry. Before the concentration and separation treatment, desalting treatment may be performed using Ziptip or the like. It is preferable to use a mixture of 3 H 4 NBA and / or 3-CH CA and 4-CH CA as a matrix for the sample for mass spectrometry.
  • the prepared sample for mass spectrometry can be used for MS measurement for quantification and MSZMS measurement for identification.
  • identification and quantification can be performed simply by preparing a single sample for mass spectrometry, and in an environment where not only an MS measurement mass spectrometer but also an MS ZMS measurement mass spectrometer can be used.
  • This is a very useful method.
  • it is possible to perform a differential display with a single gel.
  • the present invention can be said to be a very useful method.
  • the present invention also provides a chip for concentration and separation.
  • the concentration and separation chip of the present invention is an instrument that can be usefully used in the protein identification and quantification method of the present invention. More specifically, a chip for concentration and separation that is preferably used in the preferred form of the above-described protein identification and quantification method, that is, the form using the N B S reagent.
  • the instrument in which the carrier is filled or fixed is a pipette tip.
  • the material of the instrument include polypropylene resin, polyethylene resin, and fluororesin (such as PTEE, PTFE, and ET FE) that are used in commercially available pipette tips.
  • the dimensions of the device can be, for example, a volume of 5 to 1 000 L and a length of 3 to 5 cm, for example.
  • the carrier is as described in the above step ( ⁇ ) in the protein identification and quantification method of the present invention.
  • a particularly preferred form of the concentration and separation chip is one in which a carrier having a filter shape in which the surface of monolithic silica is modified with a phenyl group is fixed to a pipette chip.
  • the use conditions of concentration and separation chip of the present invention the temperature can be 15 to 40 ° C, P H 7 or less.
  • it is used by being attached to an apparatus having a pump function capable of sucking and discharging liquid, such as a micropipette, and is preferably used by being incorporated in an automation device.
  • an automation device it can be used for equipment such as Xcise (manufactured by Shimadzu Corporation).
  • suction and discharge can be performed through the carrier in the chip.
  • the elution solution can be sucked up and discharged through the carrier in the chip.
  • the present invention also provides a kit including the concentration and separation chip.
  • the kit of the present invention is a kit that can be usefully used in the identification and quantification method of the protein of the present invention. More specifically, the kit is preferably used in the preferred form of the above-described protein identification and quantification method, that is, in the form using the NBS reagent.
  • the NBS reagent ie, 2 -Nitro [ 13 C 6 ] benzenesulfuryl chloride and 2-nitro [ 12 C 6 ] benzenesulfuryl chloride are included as kit contents.
  • a TFA aqueous solution for example, about 1 V / V ⁇ 1 ⁇ 2
  • acetonitrile, or the like may be included in the kit contents.
  • the obtained sample for electrophoresis was subjected to two-dimensional electrophoresis. Specifically, IPG Ready strip (pH 5-8) gel is used (or pH 4-7 can be used), and isoelectric focusing is performed as the first dimension. SDS-PAGE was performed. After electrophoresis, G 250 staining was performed to detect protein spots.
  • Sample 1 is Pheny's sample that does not perform concentration separation by ip (Sample 1 1 a),
  • sample 1 a The sample was divided into the sample to be concentrated and separated by Phenyltip (Sample 1-b) and analyzed by the following method. [Sample 1 a]
  • Sample 1 a was desalted and purified using Ziptip. After that, MS was prepared using a matrix solution prepared with 4 mg CH CA (—Cyanol 4-hydroxycinnamic acid) in a concentration of 5 mgZm I in 0% acetonitrile — 0 ⁇ 1% TFA aqueous solution (v / v / v). The sample was eluted in a single volume and used as a sample for mass spectrometry. About this sample for mass spectrometry, MS measurement was performed with a MALDI-TO-F mass spectrometer (AXIMA-GFR, manufactured by Shimadzu Corporation), and PMF analysis was performed.
  • MALDI-TO-F MALDI-TO-F mass spectrometer
  • MA LDI -TO F MS scan Bae spectrum ( Figure 1) shows, on the one hand the peptide fragment having 13 CNBS group ions being barely detectable, from peptide fragments having the 13 CN BS group 1 6 Da and 32 Da mass numbers are also detected.
  • FIG. 1 in the range of m / z 2898-2952, ions of a peptide fragment having a 13 CNBS group, and ions having 16 Da and 32 Da mass numbers smaller than the fragment are detected. It is enlarged and displayed separately. These ions with lower mass numbers are generated by the loss of one or two nitro group oxygens in the peptide fragment with 13 CN BS groups, respectively.
  • “dividing one peak of a peptide fragment having a CNBS group into two peaks means an unfavorable result from the viewpoint of analysis sensitivity.
  • Sample 1-b was desalted and purified using Ziptip. After that, Phenyltip (filter type silica monolith is chemically bonded to phenyl group, and 10 ⁇ 1 volume pipette
  • Phenyltip filter type silica monolith is chemically bonded to phenyl group
  • 10 ⁇ 1 volume pipette The NBS group-containing peptide fragments were concentrated and separated using a fixed to a chip and obtained by custom order from Kyoto Monotech Co., Ltd. In concentrated separation, chemical species other than NBS group-containing peptide fragments are actively removed by desalting with 0.1% aqueous TFA (0.1% TFA) (v / v / v). did.
  • 4-CHCA (monocyan-4-hydroxycinnamic acid) was prepared at a concentration of 5 mgZm I in 70% acetonitrile and 0.1% TFA aqueous solution (v / v / v).
  • TFA aqueous solution
  • 3H4NBA 3-hydroxy-4-nitrobenzoic acid
  • FIG. 2 shows the obtained MALD I-T OF MS spectrum.
  • the range near m / z 2900-2960 where ions of peptide fragments having 13 CNBS groups are detected is shown separately enlarged.
  • the MA LDI-TO FMS spectrum in Fig. 2 the only peptide fragment ion with 13 CNBS groups was detected and generated from the peptide fragment as detected in Sample 1-a above.
  • One 16 Da and one 32 Da peaks were not detected. Therefore, it can be said that favorable results were obtained from the viewpoint of analysis sensitivity.
  • sample 1b since it is not necessary to perform PMF analysis on sample 1b, it was possible to perform highly sensitive analysis by using a mixed matrix.
  • a 13 CNBS group-containing peptide fragment having the sequence of KQYPIVSIEDGLDESDDGFAYQTKV (SEQ ID NO: 1) was detected as an NBS group-containing peptide fragment, but the score was 77, sample 1—a It was lower than the score.
  • the sample 1 a has a high identification score, but the analysis caused by the 16 Da and 32 Da fragments generated undesirably from the peptide fragments to be detected. The sensitivity was lowered.
  • Sample 2 is a sample that is not concentrated and separated by Phenyltip (Sample 2-a),
  • sample 2-b Then was divided into samples (sample 2-b and sample 2-c) to be concentrated and separated by Phenyltip and analyzed by the following method.
  • Sample 2—a was subjected to the same treatment as Sample 1—a.
  • Sample 2-b was subjected to the same treatment as sample 1-b.
  • Sample 2—c was treated in the same manner as Sample 1b above, except that desalting and purification using Z ⁇ pt ip was not performed.
  • Sample 3 is a sample that Pheny does not perform concentration separation with ip (Sample 3- a ),
  • the sample was divided into the sample to be concentrated and separated by Phenyltip (Sample 3-b) and analyzed by the following method.
  • Sample 3—a was subjected to the same treatment as Sample 1-a.
  • Sample 3—b was treated in the same way as Sample 1—b.
  • the EBS protein is the target of analysis, and the NBS reagent that has the ability to modify tributofan residues is used as a modification reagent for isotope labeling, and a phenyl group-containing carrier is used as a carrier for concentration and separation.
  • the present invention is also applicable to proteins other than E. coli, isotope labeling modifying reagents other than NBS reagents, and phenyl group-containing carriers.
  • the examples of these four samples are therefore merely illustrative in all respects and should not be interpreted in a limited way. Further, all modifications belonging to the equivalent scope of the claims are within the scope of the present invention.

Abstract

La présente invention concerne un procédé destiné à l'analyse d'un protéome utilisant une électrophorèse qui permet l'affichage différentiel sur un seul gel. Elle concerne un procédé pour l'identification ou la quantification d'un échantillon de protéine comprenant les étapes consistant à (i) fournir deux échantillons de protéine, (ii) modifier les deux protéines séparément par l'utilisation de réactifs de modification qui diffèrent les uns des autres uniquement par la composition isotopique, respectivement (c'est-à-dire, en marquant l'un ou l'autre des deux échantillons de protéine par le procédé de marquage avec des isotopes), (iii) mélanger les deux échantillons de protéine modifiés ensemble, (iv) réaliser une électrophorèse, (v) réaliser une fragmentation dans un gel, (vi) concentrer et séparer les peptides modifiés et (vii) réaliser une spectrométrie de masse des peptides résultants.
PCT/JP2006/306396 2006-03-22 2006-03-22 Procede d'identification et de quantification pour un echantillon de proteine a l'aide d'une electrophorese et d'une spectrometrie de masse WO2007108140A1 (fr)

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CN111587373A (zh) * 2018-01-09 2020-08-25 株式会社岛津制作所 蛋白质鉴定方法

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WO2004002950A1 (fr) * 2002-06-28 2004-01-08 Shimadzu Corporation Compose sulfenyle, reactif de marquage. et procede d'analyse de peptide
JP2004301715A (ja) * 2003-03-31 2004-10-28 Institute Of Physical & Chemical Research タンパク質の一次構造情報収集方法、それに用いる疎水性微細粒子担体及びタンパク質一次構造情報自動取得システム
JP2005189104A (ja) * 2003-12-25 2005-07-14 Shimadzu Corp タンパク質又はペプチドの濃縮分離法
JP2005326391A (ja) * 2004-04-13 2005-11-24 Shimadzu Corp Ｍａｌｄｉ質量分析装置による疎水性ペプチドの測定方法
JP2006058111A (ja) * 2004-08-19 2006-03-02 Shimadzu Corp 蛋白質同定処理方法及び装置

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Publication number Priority date Publication date Assignee Title
WO2004002950A1 (fr) * 2002-06-28 2004-01-08 Shimadzu Corporation Compose sulfenyle, reactif de marquage. et procede d'analyse de peptide
JP2004301715A (ja) * 2003-03-31 2004-10-28 Institute Of Physical & Chemical Research タンパク質の一次構造情報収集方法、それに用いる疎水性微細粒子担体及びタンパク質一次構造情報自動取得システム
JP2005189104A (ja) * 2003-12-25 2005-07-14 Shimadzu Corp タンパク質又はペプチドの濃縮分離法
JP2005326391A (ja) * 2004-04-13 2005-11-24 Shimadzu Corp Ｍａｌｄｉ質量分析装置による疎水性ペプチドの測定方法
JP2006058111A (ja) * 2004-08-19 2006-03-02 Shimadzu Corp 蛋白質同定処理方法及び装置

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111587373A (zh) * 2018-01-09 2020-08-25 株式会社岛津制作所 蛋白质鉴定方法

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