CN106153420A - Isotope dilution mass spectrometry quantitatively detects serum low-abundance protein pre-treatment flow process - Google Patents

Abstract

The invention discloses isotope dilution mass spectrometry and quantitatively detect serum low-abundance protein pre-treatment flow process, it relates to medical sanitary technology field；Its step is as follows: step one: high-abundance proteins is removed；Step 2: denaturation；Step 3: reduction；Step 4: alkylation；Step 5: terminate alkylation；Step 6: trypsinization；Step 7: SPE；Step 8: be evaporated；Step 9: redissolve；Present invention repeatability and repdocutbility are good, and broad covered area, it is adaptable to the pre-treatment of various low-abundance proteins in isotope dilution mass spectrometry detection serum.

Description

Isotope dilution mass spectrometry quantitatively detects serum low-abundance protein pre-treatment flow process

Technical field

The present invention relates to medical sanitary technology field, being specifically related to a kind of isotope dilution mass spectrometry, quantitatively to detect serum low Abundance protein pre-treatment flow process.

Background technology

Isotope dilution mass spectrometry high specificity, repdocutbility are good, are therefore the optimal of serum low-abundance protein absolute quantitation Method, but the pre-treatment that the accuracy of the method depends significantly on albumen is good and bad.Existing serum low-abundance protein pre-treatment Flow process is more, differs greatly, generally there is the defect of repeatability and repdocutbility difference, had a strong impact on isotope dilute between different flow processs Release the accurate quantitative analysis to serum low-abundance protein for the mass spectrography.

Content of the invention

Present invention aims to defect and the deficiency of prior art, provide isotope dilution mass spectrometry quantitatively to detect Serum low-abundance protein pre-treatment flow process.

In order to solve the problem existing for background technology, it is low rich that the isotope dilution mass spectrometry of the present invention quantitatively detects serum Degree albumen pre-treatment flow process, its step is as follows:

Step one: high-abundance proteins is removed: 100 μ L serum are pressed reagent specification step and usesBlue Albumin&IgG Depletion Kit removes overwhelming majority albumin and IgG, takes the blood after 180 μ L remove high-abundance proteins Clearly, and mix with the ammonium bicarbonate soln of 70 μ L 25mM；

Step 2: denaturation: first thermal denaturation 5min under 100 DEG C of water-baths by above-mentioned mixing sample, then ice bath 5min cooling, It is subsequently adding about 96.1mg urea and carries out chemical modification, stand 5min after whirlpool 30s, be eventually adding 250 μ L 25mM ammonium hydrogen carbonate Solution dilutes；

Step 3: reduction: add 50 μ L 100mM dithiothreitol (DTT)s, 60 DEG C of water-bath 30min, make albuminate open Disulfide bond keeps reducing condition；

Step 4: alkylation: add 50 μ L 550mM iodoacetamides, in dark, effect 30min under room temperature, makes cysteine On thiol alkylation, prevent the formation of disulfide bond；

Step 5: terminate alkylation: add 185 μ L 100mM dithiothreitol (DTT)s terminate alkylation, and dithiothreitol (DTT) with The mol ratio of iodoacetamide is 1: 2, acts on 30min under room temperature, prevents the excessive non-cystine residue of iodoacetamide alkylation；

Step 6: trypsinization: add 1mL 25mM ammonium bicarbonate soln dilution urea so that it is final concentration is less than 1M, so Rear addition 100 μ L 1mg/L isotopes mark peptides, add pancreatin, and the mass ratio of pancreas enzyme-to-substrate are 1: 20,37 DEG C of effects 30min, makes proteolysis become polypeptide, is eventually adding 35 μ L 5% trifluoroacetic acids and terminates digestion；

Step 7: SPE: by enzymolysis product Waters Sep-pak C18 solid-phase extraction column purification enrichment, first Activate respectively with 3mL methyl alcohol and 3mL ultra-pure water and balance pillar, being subsequently adding enzymolysis product, then clean pillar with 2mL ultra-pure water Twice, 1mL 100% acetonitrile extraction is finally used；

Step 8: be evaporated: be evaporated extraction product Nitrogen evaporator, nitrogen flow rate is 8L/min, and being evaporated temperature is 45 DEG C；

Step 9: redissolve: redissolve being evaporated the ultra-pure water containing 0.1% formic acid for the product 400 μ L.

Present invention have the beneficial effect that repeatability and repdocutbility are good, and broad covered area, it is adaptable to isotope dilution mass spectrometry is examined Survey the pre-treatment of various low-abundance proteins in serum.

Detailed description of the invention

This detailed description of the invention adopts the following technical scheme that its step is as follows:

Step one: high-abundance proteins is removed: 100 μ L serum are pressed reagent specification step and usesBlue Albumin&IgG Depletion Kit (Sigma-Aldrich) removes overwhelming majority albumin and IgG, takes 180 μ L and removes height Serum after abundance protein, and mix with ammonium hydrogen carbonate (ABC) solution of 70 μ L 25mM；

Step 2: denaturation: first thermal denaturation 5min under 100 DEG C of water-baths by above-mentioned mixing sample, then ice bath 5min cooling, It is subsequently adding about 96.1mg urea (final concentration 8M) and carries out chemical modification, stand 5min after whirlpool 30s, be eventually adding 250 μ L 25mM ABC solution dilutes；

Step 3: reduction: add 50 μ L 100mM dithiothreitol (DTT) (DTT) (final concentration 10mM), 60 DEG C of water-bath 30min, The disulfide bond making albuminate open keeps reducing condition；

Step 4: alkylation: add 50 μ L 550mM iodoacetamide (IAM) (final concentration 50mM), make under room temperature in dark With 30min, make the sulfydryl (-SH) on cysteine be alkylated, prevent the formation of disulfide bond；

Step 5: terminate alkylation: add 185 μ L 100mM DTT terminate alkylation (DTT/IAM (mol/mol)=1: 2), act on 30min under room temperature, prevent the excessive non-cystine residue of IAM alkylation；

Step 6: trypsinization: add 1mL 25mM ABC solution dilution urea so that it is final concentration is less than 1M, then adds Enter 100 μ L 1mg/L isotope mark peptides, add pancreatin (pancreatin/substrate (w/w)=1: 20) 37 DEG C effect 30min, make egg White matter is hydrolyzed into polypeptide, is eventually adding 35 μ L 5% trifluoroacetic acid (TFA) and terminates digestion；

Step 7: SPE: by enzymolysis product Waters Sep-pak C18 solid-phase extraction column purification enrichment, first Activate respectively with 3mL methyl alcohol and 3mL ultra-pure water and balance pillar, being subsequently adding enzymolysis product, then clean pillar with 2mL ultra-pure water Twice, finally with 1mL 100% acetonitrile (ACN) extraction；

Step 8: be evaporated: be evaporated extraction product Nitrogen evaporator, nitrogen flow rate is 8L/min, and being evaporated temperature is 45 DEG C；

Step 9: redissolve: the ultra-pure water containing 0.1% formic acid (FA) for the product 400 μ L will be evaporated and redissolve.

The above, only in order to technical scheme to be described and unrestricted, those of ordinary skill in the art are to this Bright technical scheme made other modification or equivalent, without departing from the spirit and scope of technical solution of the present invention, All should cover in the middle of scope of the presently claimed invention.

Claims (1)

1. isotope dilution mass spectrometry quantitatively detects serum low-abundance protein pre-treatment flow process, it is characterised in that: its step is such as Under:

Step one: high-abundance proteins is removed: 100 μ L serum are pressed reagent specification step and uses Albumin & IgG Depletion Kit removes overwhelming majority albumin and IgG, after taking 180 μ L removal high-abundance proteins Serum, and mix with the ammonium bicarbonate soln of 70 μ L 25mM；

Step 2: denaturation: first thermal denaturation 5min under 100 DEG C of water-baths by above-mentioned mixing sample, then ice bath 5min cooling, then Add about 96.1mg urea to carry out chemical modification, stand 5min after whirlpool 30s, be eventually adding 250 μ L 25mM ammonium bicarbonate solns Dilution；

Step 3: reduction: add 50 μ L 100mM dithiothreitol (DTT)s, 60 DEG C of water-bath 30min, make two sulphur that albuminate has been opened Key keeps reducing condition；

Step 4: alkylation: add 50 μ L 550mM iodoacetamides, in dark, effect 30min under room temperature, makes on cysteine Thiol alkylation, prevents the formation of disulfide bond；

Step 5: terminate alkylation: add 185 μ L 100mM dithiothreitol (DTT)s to terminate alkylation, and dithiothreitol (DTT) and iodine second The mol ratio of acid amides is 1: 2, acts on 30min under room temperature, prevents the excessive non-cystine residue of iodoacetamide alkylation；

Step 6: trypsinization: add 1mL 25mM ammonium bicarbonate soln dilution urea so that it is final concentration is less than 1M, then adds Enter 100 μ L 1mg/L isotope mark peptides, add pancreatin, and the mass ratio of pancreas enzyme-to-substrate is 1: 20,37 DEG C of effects 30min, makes proteolysis become polypeptide, is eventually adding 35 μ L 5% trifluoroacetic acids and terminates digestion；

Step 7: SPE: by enzymolysis product Waters Sep-pak C18 solid-phase extraction column purification enrichment, first use 3mL methyl alcohol and 3mL ultra-pure water activate respectively and balance pillar, are subsequently adding enzymolysis product, then clean pillar two with 2mL ultra-pure water Secondary, finally use 1mL 100% acetonitrile extraction；

Step 8: be evaporated: be evaporated extraction product Nitrogen evaporator, nitrogen flow rate is 8L/min, and being evaporated temperature is 45 DEG C；

Step 9: redissolve: redissolve being evaporated the ultra-pure water containing 0.1% formic acid for the product 400 μ L.