WO2024021228A1 - Sirt6 h133y蛋白及其富集豆蔻酰化修饰肽段的方法和应用 - Google Patents

Sirt6 h133y蛋白及其富集豆蔻酰化修饰肽段的方法和应用 Download PDF

Info

Publication number
WO2024021228A1
WO2024021228A1 PCT/CN2022/117402 CN2022117402W WO2024021228A1 WO 2024021228 A1 WO2024021228 A1 WO 2024021228A1 CN 2022117402 W CN2022117402 W CN 2022117402W WO 2024021228 A1 WO2024021228 A1 WO 2024021228A1
Authority
WO
WIPO (PCT)
Prior art keywords
sirt6
protein
flag
plasmid
seq
Prior art date
Application number
PCT/CN2022/117402
Other languages
English (en)
French (fr)
Inventor
李华
李鹏
吴娜
葛均波
Original Assignee
复旦大学附属中山医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 复旦大学附属中山医院 filed Critical 复旦大学附属中山医院
Publication of WO2024021228A1 publication Critical patent/WO2024021228A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers

Definitions

  • the invention belongs to the technical field of protein enrichment, and specifically relates to a SIRT6 H133Y protein and its method and application for enriching myristoylated modified peptide segments.
  • SIRT6 is a member of the Sirtuins family, and current research has found that it has deacetylation, mono-ADP-ribosylation, and demyristoylation activities. SIRT6 plays an important role in physiological and pathological processes, such as maintaining telomere and genome stability, DNA repair, gene expression, chronic inflammation, sugar/lipid metabolism, and cardiac hypertrophy and remodeling. Although SIRT6 is involved in multiple biological functions, the specific biochemical activities required for each function are still quite unclear. Therefore, identifying which activities regulate which biological processes is critical to understanding the molecular function of SIRT6 and accurately developing effectors or drugs targeting SIRT6.
  • SIRT6 has the function of demyristoylating lysine. They found that the demyristoylation activity of SIRT6 is almost 300 times higher than its deacetylation activity, and it can bind myristoyl groups in an amino acid sequence-independent manner. However, there is currently very little functional analysis of the demyristoylation modification of SIRT6. Due to limitations in detection technology, there is currently no good method for high-throughput identification of lysine myristoylation-modified proteins, which results in the main enzyme activity of SIRT6 (demyristoylation enzyme activity) being ignored, and lysine myristoylation Functional research on modification has stalled.
  • modified peptide enrichment mass spectrometry the protein in the test sample is enzymatically decomposed into peptide fragments, and the modified peptide fragments are obtained by enriching them through a certain method.
  • the name of the modified protein is obtained through mass spectrometry detection. and modification sites.
  • an efficient modified peptide enrichment strategy is the most critical.
  • pan-acetylation antibody enrichment strategy lysine acetylation modification has been confirmed to be a widespread modification type; through the ADP-ribose binding protein Af1521 macro domain enrichment strategy, a large number of lysine ADP ribose groups Chemically modified proteins were discovered, and it was found that the occurrence of such modifications may be related to oxidative stress.
  • the purpose of the present invention is to provide a SIRT6 H133Y protein and its method and application for enriching myristoylated modified peptides. Since SIRT6 itself can bind myristoyl groups, a method was established to enrich myristoylation-modified peptides using SIRT6 H133Y mutant protein, and mass spectrometry identification of lysine myristoylation-modified proteomics was proposed.
  • the invention provides a method for preparing SIRT6 H133Y protein, which includes:
  • SIRT6-FLAG fragment Use chemically synthesized SIRT6-FLAG (the SIRT6-FLAG gene was synthesized by Qingke Biotech (China)) as a template to obtain the SIRT6-FLAG fragment through SIRT6 primers.
  • Synthetic plasmid fragment Using pET28a plasmid as template, the plasmid fragment is amplified by pET28a primer.
  • the expression plasmid pET28TEV-SIRT6 was obtained by homologous splicing of the SIRT6-FLAG fragment and the plasmid fragment; using pET28TEV-SIRT6 as a template, PCR site-directed mutagenesis technology was performed using H133Y primers (that is, H133Y was carried out on the SIRT6 gene in pET28TEV-SIRT6 through PCR site-directed mutagenesis technology point mutation), amplify the overexpression plasmid pET28TEV-SIRT6H133Y, transform the overexpression plasmid pET28TEV-SIRT6H133Y into E. coli, and within 2-3 days, sufficient SIRT6 H133Y protein can be purified through the E. coli protein expression system and His tag.
  • the base sequence of the SIRT6-FLAG fragment (SEQ ID NO.1) is:
  • amino acid sequence SEQ ID NO.2 of the HIS tag and Flag tag double tag SIRT6 H133Y protein (the underline highlights the H133Y mutation site):
  • pET28a primers are:
  • pET28a-F gcggccgctttcgaatctagagc(SEQ ID NO.3);
  • pET28a-R gaattccggatccatggcgccctg(SEQ ID NO.4);
  • the SIRT6 primers are:
  • SIRT6-F gcgccatggatccggaattcatgagcgttaactatg(SEQ ID NO.5);
  • SIRT6-R ctagattcgaaagcggccgctttatttatcatcatcatc (SEQ ID NO. 6).
  • the primers for PCR site-directed mutation amplification are:
  • H133Y-F cagaactgTATggtaatatgtttgttgaag(SEQ ID NO.7);
  • H133Y-R catattaccATAcagttctgccagtttatc(SEQ ID NO.8).
  • the present invention provides a SIRT6 H133Y protein, which is obtained by the above-mentioned preparation method.
  • the present invention provides a method for enriching SIRT6 H133Y protein with myristoylated modified peptides using the above-mentioned SIRT6 H133Y protein, which includes the following steps:
  • Cell samples Cells can be cultured by adding inhibitors and lipids to DMEM low-sugar medium, or adding myristic acid analogues to DMEM low-sugar medium to increase the level of myristoylation modification of intracellular proteins and collect them. Cells can be digested and lysed to extract full protein; tissue samples: Tissues can be directly digested and lysed to extract full protein. The extracted protein is added to trypsin for enzymatic digestion to obtain enzymatic peptide fragments, which are then freeze-dried for later use;
  • the inhibitor is a carnitine palmitoyltransferase 1a inhibitor (CPT1 ⁇ inhibitor, Aladdin Company, CAS number: 124083-20-1), and the lipid is a lipid concentrate with clear chemical composition (Chemistry The lipid concentrate with clear composition comes from Thermo Company, for details, please see: Thermo official website www.thermofisher.cn/cn/zh/home/technical-resources/media-formulation.249.html).
  • the myristic acid analogue is palmitic acid acetylene. (ALK14) (Click Chemistry Tools, CAS number: 99208-90-9).
  • step (1) the cell culture temperature is 37°C and the time is 6-12 hours.
  • tissue samples generally refer to tissue samples, including various human tissues and various animal tissues.
  • step (1) the enzyme digestion temperature is 37°C and the time is 16-18h.
  • the buffer is Myr-IP buffer, which includes phosphate buffered saline solution, 0.2% Tween-20 and 20% acetonitrile.
  • step (2) the incubation temperature is 4°C and the time is 16-18 hours; or room temperature is 2-4 hours.
  • the elution buffer includes 0.2 mol/L glycine and 20% acetonitrile.
  • step (5) the incubation temperature is room temperature or 37°C, and the incubation time is 5-10 minutes.
  • step (5) the centrifugation speed is 12000 rpm and the centrifugation time is 15-30 min.
  • the present invention provides a myristoylated modified peptide segment, which is obtained by the above method.
  • the present invention provides an application of the above-mentioned myristoylated modified peptide segment.
  • the myristoylated modified peptide segment can be used in mass spectrometry identification to identify myristoylated modified proteins and modification sites.
  • the present invention is based on the binding ability of SIRT6 H133Y to myristoyl, and co-immunoprecipitates the myristoylated modified peptides through the His tag purified protein and Flag tag on the SIRT6 H133Y protein, thereby enriching and obtaining the myristoylated modification.
  • Peptide segment; mass spectrometry is performed on the myristoylated modified peptide segment, and the myristoylated modified target protein and modification site are analyzed. Therefore, the present invention is to find potential myristoylated modified target protein in the organism, identify the modification site, and provide analysis Provide methodological support for the physiological significance of lysine myristoylation modification.
  • the method of the present invention can not only identify lysine myristoylation modification, but also identify other long-chain fatty acylation modification sites (such as palmitoylation and palmitoylation) through different mass shifts.
  • the enrichment method of the present invention is low-cost and short-time consuming, and the enrichment material SIRT6 H133Y can be prepared in large quantities in a short time through the conventional E. coli protein expression system. Anti-Flag magnetic beads are easily available.
  • Figure 1 is a schematic workflow diagram of the proteomic analysis of myristoylation modification using peptide enrichment mass spectrometry in the present invention.
  • Figure 2 is a quality inspection diagram of the HIS tag and Flag tag dual-label SIRT6 H133Y purified protein process in Example 1 of the present invention.
  • Figure 3 is a verification diagram of SIRT6 knockdown 293T cells in Example 2 of the present invention.
  • Figure 4 is a diagram showing the myristoylation level of the whole protein after cells were cultured in DMEM low-sugar medium with carnitine palmitoyltransferase 1a inhibitor and lipid concentrate with clear chemical composition in Example 3 of the present invention.
  • Figure 5 is a quality inspection chart of protein extraction from SIRT6 knockdown 293T cells in Example 3 of the present invention.
  • Figure 6 is a liquid chromatography peptide separation diagram of a 293T cell sample cultured with ALK14 in Example 5 of the present invention.
  • Figure 7 is a mass spectrum of the lysine myristoylated modified peptide in Example 5 of the present invention.
  • the invention provides a SIRT6 H133Y protein and a method and application for enriching myristoylated modified peptide segments.
  • the cell samples were protein extracted, proteolytically hydrolyzed, and myristoylated modified peptides were enriched by dSIRT6.
  • the myristoylated modified peptides were analyzed by mass spectrometry to obtain the myristoylated proteome profile. That is, a method of protecting and utilizing the SIRT6 H133Y protein with His tag and Flag tag dual tags to enrich myristoylated modified peptides and conducting mass spectrometry to identify the myristoylated modified proteins.
  • the specific description is as follows:
  • the expression vector of the protein mutant SIRT6 H133Y was designed through molecular biology methods.
  • the SIRT6-FLAG fragment was obtained by using SIRT6-FLAG as the template and the SIRT6 primer.
  • the plasmid fragment was amplified by using the pET28a plasmid as the template and the pET28a primer.
  • the SIRT6-FLAG fragment and the plasmid fragment were homologously spliced by seamless cloning technology to obtain the expression plasmid pET28TEV.
  • -SIRT6; SIRT6 has a His tag at the N end and a Flag tag at the C end.
  • the base sequence of the SIRT6-FLAG fragment (SEQ ID NO.1) is:
  • pET28a primers are:
  • pET28a-F gcggccgctttcgaatctagagc(SEQ ID NO.3);
  • pET28a-R gaattccggatccatggcgccctg (SEQ ID NO.4).
  • the SIRT6 primers are:
  • SIRT6-F gcgccatggatccggaattcatgagcgttaactatg(SEQ ID NO.5);
  • SIRT6-R ctagattcgaaagcggccgctttatttatcatcatcatc (SEQ ID NO. 6).
  • the primers for PCR site-directed mutagenesis amplification are:
  • H133Y-F cagaactgTATggtaatatgtttgttgaag(SEQ ID NO.7);
  • H133Y-R catattaccATAcagttctgccagtttatc(SEQ ID NO.8).
  • Etomoxir CPT1 ⁇ inhibitor
  • CPT1 ⁇ inhibitor can be injected into animals or added to the culture medium to inhibit carnitine acyltransferase 1, thereby increasing the content of long-chain fatty acyl-CoA in the cytoplasm, which is beneficial to improving myristoylation.
  • knocking out the SIRT6 gene is also beneficial to increasing the abundance of myristoylation modification.
  • step (3) Mix the reaction solution in step (1) with the pretreated Anti-Flag magnetic beads (the magnetic beads can be purchased from the domestic Beenbio company (Cat. No. PR002) and the foreign Thermo Scientific company (Cat. No. A36797)). Enrichment reaction, incubate at 4°C for 16-18h;
  • Myr-IP buffer 1X PBS (without Ca 2+ , Mg 2+ , pH value 7.4), 0.2% Tween-20, 20% acetonitrile; elution buffer: 0.2mol/L glycine, 20% acetonitrile, The pH value is 2.2.
  • Mass spectrometry samples were separated using a high-performance liquid phase system EASY-nLC 1200 with nanoliter flow rate.
  • Mobile phase A is a 0.1% formic acid aqueous solution
  • mobile phase B is a 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 80%).
  • the chromatographic column is equilibrated with 100% mobile phase A, and then the enzymatic peptide fragments of the sample are transported to the loading column (2cm, ID100 ⁇ m, 3 ⁇ m, C18) by the automatic sampler, and then pass through the analytical column (15cm, ID150 ⁇ m, 1.9 ⁇ m, C18) for separation, with a flow rate of 600 nL/min.
  • the relevant liquid gradients are as follows: 75min gradient: 0-5min, liquid linear gradient from 4-20%; 5-65min, liquid B linear gradient from 20-50%; 66-75min, liquid B maintained at 100%.
  • the peptide samples were separated by the analytical chromatography column, and the HPLC peptide separation chart proved that the peptide separation effect was good and the abundance was good, and then the Q Exactive HF-X mass spectrometer was used for mass spectrometry detection.
  • the detection method is positive ion mode
  • the precursor ion scanning range is 300-1400m/z
  • the first-level mass spectrometry resolution is 120,000at 200m/z
  • the AGC (Automatic gain control) target is 3e6
  • the Maximum IT is 30ms
  • the dynamic exclusion time (Dynamic exclusion time) ) is 12.0s.
  • the mass-to-charge ratios of peptides and peptide fragments were collected according to the following method: 60 fragment spectra (MS2scan) were collected after each full scan (full scan), using HCD fragmentation mode, Normalized Collision Energy was 27%, and Isolation window was 1.6m/ z, secondary mass spectrometry resolution 7,500at 200m/z.
  • the raw data (RAW files) collected by mass spectrometry analysis are searched in the database through Thermo Proteome Discoverer software, and the identification information of the protein sample is finally obtained.
  • the search parameters are set as follows: enzyme is Trypsin; missed cleavage sites are set to 2; non-ALK14 samples are added, and dynamic modification settings include Oxidation (M) and Myr-(K) and Myr-(G) (mass migration 210.356Da; ALK14 is added to the sample, and the Oxidation (M) and ALK14-(K) and ALK14-(G) are dynamically modified (mass shift 234.377Da).
  • the proteins identified by the database search must pass the set filtering parameter FDR ⁇ 0.01.
  • the Mass spectra of myristoylated peptides and lysine myristoylated proteins are set as follows: enzyme is Trypsin; missed cleavage sites are set to 2; non-ALK14 samples are added, and dynamic modification settings include Ox
  • the SIRT6-FLAG fragment was synthesized using SIRT6-FLAG as a template and SIRT6-F/R primers.
  • the plasmid fragment was amplified using pET28a plasmid as a template and pET28a-F/R primers.
  • the SIRT6-FLAG fragment and plasmid were combined using seamless cloning technology.
  • the fragments were spliced through homologous splicing to obtain the expression plasmid pET28TEV-SIRT6, which has a His tag at the N end and a Flag tag at the C end.
  • PCR site-directed mutation was performed with primers H133Y-F/R to construct an overexpression plasmid of SIRT6 H133Y with HIS tag and Flag tag (pET28TEV-SIRT6H133Y).
  • the base sequence of the SIRT6-FLAG fragment (SEQ ID NO.1) is:
  • pET28a-F gcggccgctttcgaatctagagc(SEQ ID NO.3);
  • pET28a-R gaattccggatccatggcgccctg(SEQ ID NO.4);
  • SIRT6-F gcgccatggatccggaattcatgagcgttaactatg(SEQ ID NO.5);
  • SIRT6-R ctagattcgaaagcggccgctttatttatcatcatc(SEQ ID NO.6);
  • H133Y-F cagaactgTATggtaatatgtttgttgaag(SEQ ID NO.7);
  • H133Y-R catattaccATAcagttctgccagtttatc(SEQ ID NO.8).
  • pET28TEV-SIRT6H133Y is transformed into E. coli ArcticExpress (ED3).
  • the transformant is grown in 2X YT medium (kanamycin 50 ⁇ g/mL, gentamicin 40 ⁇ g/mL). When OD 600 reaches 0.8, add 0.2mmol/L IPTG. Protein expression was induced overnight at 16°C.
  • the protein was dialyzed using dialysis buffer (50mmol/L Tris-HCl, 250mmol/L NaCl and 10% glycerol, pH value 7.2) to remove imidazole, and the obtained protein was stored at -80°C.
  • dialysis buffer 50mmol/L Tris-HCl, 250mmol/L NaCl and 10% glycerol, pH value 7.2
  • SIRT6 H133Y protein (SEQ ID NO.2) (the underline highlights the H133Y mutation site):
  • 293T cells were transfected with SIRT6shRNA virus, and Polybrene was added to increase the infection efficiency.
  • the medium was changed after 6 hours of transfection (use high-sugar DMEM medium). Puromycin was used for positive selection 48 hours after transfection, and a cell line stably expressing SIRT6 shRNA was obtained.
  • Western blotting results demonstrated that 293T SIRT6 knockdown efficiency was good (Figure 3).
  • SIRT6 knockdown 293T cells were cultured in high-glucose DMEM medium containing 10% FBS. After the cell confluence rate reached about 80%, they were cultured in low-glucose (1g/mL) DMEM, in which Etomoxir (cpt1a inhibitor) was added. agent, China Aladdin Company) 50 ⁇ mol/L, 1% lipid concentrate with clear chemical composition, and collected samples after 12 hours of culture. Western blotting results proved that the above culture method can increase the abundance of myristoylation modification ( Figure 4). Cells can also be added with 10 ⁇ g/mL ALK14 (myristic acid analog) through the culture medium, and collected after 12 hours of culture.
  • ALK14 myristic acid analog
  • step (3) Mix the reaction solution in step (1) with the pretreated Anti-Flag magnetic beads for enrichment reaction, and incubate at 4°C for 18 hours;
  • Myr-IP buffer 1X PBS (without Ca 2+ , Mg 2+ , pH 7.4), 0.2% Tween-20, 20% acetonitrile.
  • Elution buffer 0.2mol/L glycine, 20% acetonitrile, pH value 2.2.
  • the samples were separated using a high-performance liquid phase system EASY-nLC 1200 with nanoliter flow rate.
  • Mobile phase A is a 0.1% formic acid aqueous solution
  • mobile phase B is a 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 80%).
  • the chromatographic column is equilibrated with 100% mobile phase A, and then the enzymatic peptide fragments of the sample are transported to the loading column (2cm, ID100 ⁇ m, 3 ⁇ m, C18) by the automatic sampler, and then pass through the analytical column (15cm, ID150 ⁇ m, 1.9 ⁇ m, C18) for separation, with a flow rate of 600 nL/min.
  • the peptide sample was separated by the analytical chromatography column.
  • the HPLC peptide separation chart showed that the peptide separation effect was good and the abundance was good ( Figure 6). It was then detected with the Q Exactive HF-X mass spectrometer.
  • the detection method is positive ion mode, the precursor ion scan is 700m/z, the primary mass spectrometry resolution is 120,000at 200m/z, the AGC (Automatic gain control) target is 3e6, the Maximum IT is 30ms, and the dynamic exclusion time (Dynamic exclusion) is 12.0s.
  • the mass-to-charge ratio of peptides and peptide fragments was collected according to the following method: 60 fragment spectra (MS2scan) were collected after each full scan (full scan), using HCD fragmentation mode, Normalized Collision Energy was 27%, and Isolation window was 1.6m/ z, secondary mass spectrometry resolution 7,500at 200m/z.
  • the raw data (RAW files) collected by mass spectrometry analysis are searched in the database through Thermo Proteome Discoverer software, and the identification information of the protein sample is finally obtained.
  • the search parameters are set as follows: enzyme is Trypsin; missed cleavage sites are set to 2; non-ALK14 samples are added, and Oxidation (M), Myr-(K) and Myr-(G) are set for dynamic modification (mass migration 210.356Da) ; ALK14 is added to the sample, and dynamically modified to set Oxidation (M) and ALK14-(K) and ALK14-(G) (mass migration 234.377Da).
  • the target protein modified by lysine myristoylation and its modification site were obtained.
  • DUX4L3 and GLIS2 were two myristoylation-modified proteins identified among the modified proteins ( Figure 7).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明提供了一种SIRT6 H133Y蛋白及其富集豆蔻酰化修饰肽段的方法和应用，蛋白制备方法包括：以SIRT6-FLAG为模板得到SIRT6-FLAG片段，以pET28a质粒为模板得到质粒片段，两片段拼接得到表达质粒pET28TEV-SIRT6，以其为模板进行PCR定点突变技术，得到过表达质粒pET28TEV-SIRT6H133Y，转入大肠杆菌内纯化蛋白；本发明基于SIRT6H133Y蛋白对豆蔻酰基的结合力，并通过SIRT6 H133Y蛋白上的His-tag和Flag-tag标签对豆蔻酰化修饰肽段进行免疫共沉淀，富集得到豆蔻酰化修饰肽段，质谱检测分析得到豆蔻酰化修饰靶蛋白和修饰位点。

Description

SIRT6 H133Y蛋白及其富集豆蔻酰化修饰肽段的方法和应用 技术领域
本发明属于蛋白富集技术领域，具体涉及一种SIRT6 H133Y蛋白及其富集豆蔻酰化修饰肽段的方法和应用。
背景技术
SIRT6是Sirtuins家族的一员，目前研究发现其具有去乙酰化、单ADP-核糖基化、去豆蔻酰化活力。SIRT6在生理病理过程中扮演着重要角色，如维持端粒和基因组稳定、DNA修复、基因表达，慢性炎症、糖/脂代谢以及心脏肥厚和重构。SIRT6虽涉及多种生物学功能，但每种功能所需的具体生化活性仍相当模糊。因此，鉴定哪些活性调控哪种生物学过程，对于理解SIRT6的分子功能以及精准开发针对SIRT6的效应因子或药物来说至关重要。
近年来，研究者发现SIRT6具有去赖氨酸豆蔻酰化功能，发现SIRT6的去豆蔻酰化活力比其去乙酰化活力几乎高300倍，并可以非氨基酸序列依赖的结合豆蔻酰基。然而目前对于SIRT6去豆蔻酰化修饰的功能解析非常少。由于检测技术限制，目前并没有很好的方法用于高通量鉴定赖氨酸豆蔻酰化修饰蛋白，这导致SIRT6主要酶活(去豆蔻酰化酶活力)被忽视，赖氨酸豆蔻酰化修饰的功能研究停滞不前。
目前鉴定蛋白翻译后修饰主要通过修饰肽段富集质谱的方法进行，即将检测样品中的蛋白质酶解为肽段，通过一定方法富集得到修饰的肽段，最后通过质谱检测得到修饰蛋白质的名称及修饰位点。对于修饰蛋白的鉴定，高效的修饰肽段富集策略最为关键。例如通过泛乙酰化抗体富集策略，目前赖氨酸乙酰化修饰已被证实为一种广泛存在的修饰类型；通过ADP-核糖结合蛋白Af1521 macro domain富集策略，大量的赖氨酸ADP核糖基化修饰蛋白被发现，并发现这类修饰的出现可能与氧化应激相关。然而，目前并没有高效的赖氨酸豆蔻酰化修饰肽段的富集方法。
发明内容
针对现有技术中的不足，本发明的目的是提供一种SIRT6 H133Y蛋白及其富集豆蔻酰化修饰肽段的方法和应用。由于SIRT6自身可以结合豆蔻酰基，因此建立了以SIRT6 H133Y突变蛋白富集豆蔻酰化修饰肽段的方法，并提出赖氨酸豆蔻酰化修饰蛋白组学的质谱鉴定。
为达到上述目的，本发明的解决方案是：
第一方面，本发明提供了一种SIRT6 H133Y蛋白的制备方法，其包括：
化学合成SIRT6-FLAG片段：以化学合成的SIRT6-FLAG(SIRT6-FLAG基因由擎科生物(中国)的基因合成得到)为模板通过SIRT6引物得到SIRT6-FLAG片段，
合成质粒片段：以pET28a质粒为模板通过pET28a引物扩增得到质粒片段，
SIRT6-FLAG片段和质粒片段通过同源拼接得到表达质粒pET28TEV-SIRT6；以pET28TEV-SIRT6为模板，通过H133Y引物进行PCR定点突变技术(即通过PCR定点突变技术对pET28TEV-SIRT6中的SIRT6基因进行H133Y点突变)，扩增得到过表达质粒pET28TEV-SIRT6H133Y，将过表达质粒pET28TEV-SIRT6H133Y转化大肠杆菌内，2-3天内能够通过大肠杆菌蛋白质表达系统和His标签纯化得到足够的SIRT6 H133Y蛋白。
其中，SIRT6-FLAG片段的碱基序列(SEQ ID NO.1)为：
Figure PCTCN2022117402-appb-000001
HIS tag、Flag tag双标签SIRT6 H133Y蛋白的氨基酸序列(SEQ ID NO.2)(下换线突出为H133Y突变位点)：
Figure PCTCN2022117402-appb-000002
进一步地，pET28a引物分别为：
pET28a-F:gcggccgctttcgaatctagagc(SEQ ID NO.3)；
pET28a-R:gaattccggatccatggcgccctg(SEQ ID NO.4)；
SIRT6引物分别为：
SIRT6-F:gcgccatggatccggaattcatgagcgttaactatg(SEQ ID NO.5)；
SIRT6-R:ctagattcgaaagcggccgcttatttatcatcatcatc(SEQ ID NO.6)。
进一步地，得到过表达质粒pET28TEV-SIRT6H133Y时，PCR定点突变扩增的引物分别为：
H133Y-F:cagaactgTATggtaatatgtttgttgaag(SEQ ID NO.7)；
H133Y-R:catattaccATAcagttctgccagtttatc(SEQ ID NO.8)。
第二方面，本发明提供了一种SIRT6 H133Y蛋白，其由上述的制备方法得到。
第三方面，本发明提供了一种利用上述的SIRT6 H133Y蛋白进行SIRT6 H133Y蛋白富集豆蔻酰化修饰肽段的方法，其包括如下步骤：
(1)、细胞样品：细胞可以在DMEM低糖培养基内添加抑制剂和脂质、或在DMEM低糖培养基添加豆蔻酸类似物进行细胞培养，用于提高细胞内蛋白质豆蔻酰化修饰水平，收集细胞并消化裂解提取全蛋白；组织样本：组织可直接消化裂解提取全蛋白。提取的蛋白加入胰蛋白酶进行酶切，得到酶解肽段，冷干备用；
(2)、将酶解肽段溶于缓冲液中，加入SIRT6 H133Y蛋白和乙腈混合，得到第一混合液；再将Anti-Flag磁珠加入缓冲液内，然后加入第一混合液进行富集反应，孵育，得到第二混合液；
(3)、将第二混合液磁力沉淀，弃去液体，加入缓冲液洗涤，磁力沉淀，反复洗涤后，彻底弃去液体；
(4)、洗涤后的Anti-Flag磁珠加入洗脱缓冲液，孵育，洗脱，得到肽段洗脱液；
(5)、在肽段洗脱液中加入三氟乙酸进行孵育，离心，取上清液冻干，获得豆蔻酰化修饰肽段。
进一步地，步骤(1)中，抑制剂为肉碱棕榈酰转移酶1a抑制剂(CPT1αinhibitor,Aladdin公司，CAS number:124083-20-1)，脂质为化学成分明确的脂质浓缩液(化学成分明确的脂质浓缩液来源于Thermo公司，详见:Thermo官网www.thermofisher.cn/cn/zh/home/technical-resources/media-formulation.249.html)，豆蔻酸类似物为棕榈酸炔(ALK14)(Click Chemistry Tools公司，CAS number:99208-90-9)。
进一步地，步骤(1)中，细胞培养的温度为37℃，时间为6-12h。
进一步地，步骤(1)中，组织样品泛指组织样品，包括人的各种组织和动物的各种组织。
进一步地，步骤(1)中，酶切的温度为37℃，时间为16-18h。
进一步地，步骤(2)和步骤(3)中，缓冲液为Myr-IP缓冲液，其包括磷酸缓冲盐溶液、 0.2％吐温-20和20％乙腈。
进一步地，步骤(2)中，孵育的温度为4℃，时间为16-18h；或室温2-4h。
进一步地，步骤(4)中，洗脱缓冲液包括0.2mol/L甘氨酸和20％乙腈。
进一步地，步骤(5)中，孵育的温度为室温或37℃，时间为5-10min。
进一步地，步骤(5)中，离心的转速为12000rpm，时间为15-30min。
第四方面，本发明提供了豆蔻酰化修饰肽段，其由上述的方法得到。
第五方面，本发明提供了一种上述的豆蔻酰化修饰肽段的应用，该豆蔻酰化修饰肽段在质谱鉴定中得以应用，鉴定豆蔻酰化修饰蛋白及修饰位点。
由于采用上述方案，本发明的有益效果是：
第一、本发明基于SIRT6 H133Y对豆蔻酰基的结合能力，并通过SIRT6 H133Y蛋白上的His tag纯化蛋白和Flag tag标签对豆蔻酰化修饰肽段进行免疫共沉淀，从而富集得到豆蔻酰化修饰肽段；对豆蔻酰化修饰肽段进行质谱检测，分析得到豆蔻酰化修饰靶蛋白和修饰位点，故本发明为寻找生物体内潜在的豆蔻酰化修饰靶蛋白，鉴定修饰位点，为解析赖氨酸豆蔻酰化修饰的生理意义提供方法学支撑。同时本发明的方法不仅能够鉴定赖氨酸豆蔻酰化修饰，还可以通过不同的质量迁移鉴定其他长链脂肪酰化修饰位点(例如棕榈酰化和软脂酰化)。
第二、相比修饰基团泛抗体富集质谱策略，本发明的富集方法成本低，耗时短，且富集材料SIRT6 H133Y可以通过常规大肠杆菌蛋白表达系统短时间大量制备，富集所用Anti-Flag磁珠容易获得。
附图说明
图1为本发明的肽段富集质谱鉴定豆蔻酰化修饰蛋白质组学分析的工作流程示意图。
图2为本发明的实施例1中HIS tag、Flag tag双标签SIRT6 H133Y纯化蛋白流程质检图。
图3为本发明的实施例2中SIRT6敲低293T细胞验证图。
图4为本发明的实施例3中DMEM低糖培养基添加肉碱棕榈酰转移酶1a抑制剂和化学成分明确的脂质浓缩液培养细胞后全蛋白豆蔻酰化水平图。
图5为本发明的实施例3中SIRT6敲低293T细胞蛋白质提取质检图。
图6为本发明的实施例5中添加ALK14培养的293T细胞样本液相色谱肽段分离图。
图7为本发明的实施例5中赖氨酸豆蔻酰化修饰肽段质谱图。
具体实施方式
本发明提供了一种SIRT6 H133Y蛋白及其富集豆蔻酰化修饰肽段的方法和应用。
如图1所示，细胞样本经过蛋白提取，蛋白酶解后通过dSIRT6富集豆蔻酰化修饰肽段，上机质谱分析，得到豆蔻酰化修饰蛋白质组谱。即保护利用带有His tag、Flag tag双标签的 SIRT6 H133Y蛋白富集豆蔻酰化修饰肽段并进行质谱鉴定豆蔻酰化修饰蛋白的方法。具体描述如下：
富集材料SIRT6 H133Y的制备：
通过分子生物学方法设计蛋白突变体SIRT6 H133Y的表达载体。以SIRT6-FLAG为模板通过SIRT6引物得到SIRT6-FLAG片段，以pET28a质粒为模板通过pET28a引物扩增得到质粒片段，通过无缝克隆技术将SIRT6-FLAG片段和质粒片段通过同源拼接得到表达质粒pET28TEV-SIRT6；SIRT6的N端带His tag，C端带Flag tag。以pET28TEV-SIRT6为模板通过H133Y引物进行PCR定点突变技术，构建带有His tag、Flag tag双标签的SIRT6 H133Y的过表达质粒(pET28TEV-SIRT6 H133Y)。在大肠杆菌蛋白质表达系统表达N端His tag和C端Flag tag双标签的SIRT6 H133Y蛋白。利用His标签通过镍离子交换柱纯化SIRT6 H133Y蛋白，通过SDS-PAGE电泳确认纯化成功且纯度合格。蛋白经过透析后，蛋白保存于-80℃。
其中，SIRT6-FLAG片段的碱基序列(SEQ ID NO.1)为：
Figure PCTCN2022117402-appb-000003
HIS tag、Flag tag双标签SIRT6 H133Y蛋白的氨基酸序列(SEQ ID NO.2)(下换线突出为H133Y突变位点)：
Figure PCTCN2022117402-appb-000004
具体地，pET28a引物分别为：
pET28a-F:gcggccgctttcgaatctagagc(SEQ ID NO.3)；
pET28a-R:gaattccggatccatggcgccctg(SEQ ID NO.4)。
SIRT6引物分别为：
SIRT6-F:gcgccatggatccggaattcatgagcgttaactatg(SEQ ID NO.5)；
SIRT6-R:ctagattcgaaagcggccgcttatttatcatcatcatc(SEQ ID NO.6)。
PCR定点突变扩增的引物分别为：
H133Y-F:cagaactgTATggtaatatgtttgttgaag(SEQ ID NO.7)；
H133Y-R:catattaccATAcagttctgccagtttatc(SEQ ID NO.8)。
酶解肽段的制备：
由于豆蔻酰化修饰的丰度很低，可以通过动物注射或者培养基添加Etomoxir(CPT1α抑制剂)抑制肉碱脂酰转移酶1，从而提高细胞质的长链脂酰辅酶A含量，有利于提高豆蔻酰化修饰丰度。同样敲除SIRT6基因也有利于豆蔻酰化修饰丰度的提高。
Western Blotting证明细胞通过低糖(1g/mL)的DMEM培养基，加入50μmol/L的Etomoxir、1％的化学成分明确的脂质浓缩液(美国gibco)能够较好地提高豆蔻酰化修饰丰度。还可以通过培养基添加豆蔻酸类似物ALK14(棕榈酸炔)，提高模拟豆蔻酰化修饰的丰度。
收集的组织或细胞进行消化裂解后，加入8mol/L的UA裂解液，冰上超声裂解(100W，工作10s，间歇10s，循环10次)，14000rpm离心30min后取上清得到蛋白裂解液测量浓度。取样品蛋白质20μg，加入5X上样缓冲液，沸水浴5min，进行12.5％SDS-PAGE电泳(120V，75min)，考马斯亮蓝染色鉴定蛋白质提取质量。取2mg蛋白样品加入10mmol/L二硫苏糖醇(DTT)在37℃孵育2h以破坏蛋白质二硫键，后冷却至室温，加入IAA至终浓度50mmol/L，避光孵育30min用来修饰蛋白质SH基团。加入5倍体积水，将UA裂解液浓度稀释至1.5mol/L，按照50:1比例加入Trypsin，37℃酶切18h左右。通过SPE C18柱脱盐，冻干，得到酶解肽段，存放于-80℃备用。
豆蔻酰化修饰肽段的富集：
(1)酶解肽段(1mg)复溶于400μL预冷Myr-IP缓冲液中，随后加入80μL的SIRT6 H133Y蛋白(1mg/mL)和20μL乙腈，混匀，30℃反应4h，使SIRT6结合修饰肽段；
(2)取50μL的Anti-Flag磁珠于1.5mL的EP管中，加入500μL的Myr-IP缓冲液洗涤，混匀后将EP管放在磁力架上分离beads和液体，弃去液体，洗涤3次；
(3)将步骤(1)中的反应液与预处理好的Anti-Flag磁珠(该磁珠可以从国内Beenbio公司(货号PR002)和国外Thermo Scientific公司(货号A36797)购买得到)混匀进行富集反应，4℃孵育16-18h；
(4)将上述反应液从4℃取出，放在磁力架上分离，弃去液体；
(5)加入500μL的Myr-IP缓冲液洗涤，混匀后将EP管放在磁力架上分离beads和液体，弃去液体，重复洗涤3次；
(6)加入150μL洗脱缓冲液混匀，室温摇床孵育15min，放在磁力架上，将液体转入干净的1.5mL的EP管中，洗脱3次，混合；
(7)在洗脱产物中加入4％的TFA，37℃孵育10min后，12000rpm离心15min沉淀SIRT6蛋白，上清保留，可以直接作为质谱样品进行质谱鉴定或冷冻干燥保存。如果是冻干肽段用0.1％甲酸20％乙腈水溶液复溶作为质谱样品。
Myr-IP缓冲液：1X PBS(不含Ca 2+、Mg 2+，pH值为7.4)，0.2％Tween-20，20％乙腈；洗脱缓冲液：0.2mol/L甘氨酸，20％乙腈，pH值为2.2。
质谱鉴定：
质谱样品采用纳升流速的高效液相系统EASY-nLC 1200进行分离。流动相A为0.1％甲酸水溶液，流动相B为0.1％甲酸乙腈水溶液(乙腈为80％)。首先色谱柱以100％的流动相A进行平衡，随后样品的酶解肽段由自动进样器输送到上样柱(2cm，ID100μm，3μm，C18)，之后再经过分析柱(15cm，ID150μm，1.9μm，C18)进行分离，流速为600nL/min。相关液相梯度如下：75min梯度:0-5min，液线性梯度从4-20％；5-65min，B液线性梯度从20-50％；66-75min，B液维持在100％。肽段样品经分析色谱柱分离，并通过HPLC肽段分离图证明肽段分离效果良好且丰度良好，后用Q Exactive HF-X质谱仪进行质谱检测。检测方式为正离子模式，母离子扫描范围300-1400m/z，一级质谱分辨率为120,000at 200m/z，AGC(Automatic gain control)target为3e6，Maximum IT为30ms，动态排除时间(Dynamic exclusion)为12.0s。多肽和多肽碎片的质荷比按照下列方法采集：每次全扫描(full scan)后采集60个碎片图谱(MS2scan)，使用HCD碎裂模式，Normalized Collision Energy为27％，Isolation window为1.6m/z，二级质谱分辨率7,500at 200m/z。
质谱分析采集的原始数据(RAW文件)，通过Thermo Proteome Discoverer软件进行数据库检索，最终获得蛋白质样品的鉴定信息。其中搜库参数设定如下：enzyme为Trypsin；missed cleavage sites设为2；非ALK14添加样品，动态修饰设定Oxidation(M)和Myr-(K)和Myr-(G)(质量迁移210.356Da；ALK14添加样品，动态修饰设定Oxidation(M)和ALK14-(K)和ALK14-(G)(质量迁移234.377Da)。数据库检索鉴定到的蛋白质必须通过设定的过滤参数FDR≤0.01。最终获得豆蔻酰化修饰肽段质谱图和赖氨酸豆蔻酰化修饰蛋白。
下面结合附图和实施例对本发明的技术内容做进一步的说明。下述实施例是说明性的，不是限定性的，不能以下述实施例来限定本发明的保护范围。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：
大肠杆菌表达纯化SIRT6 H133Y蛋白
(1)构建带有HIS tag、Flag tag双标签的SIRT6 H133Y的过表达质粒(pET28TEV-SIRT6H133Y)：
以SIRT6-FLAG为模板通过SIRT6-F/R引物合成得到SIRT6-FLAG片段，以pET28a质粒为模板通过pET28a-F/R引物扩增得到质粒片段，通过无缝克隆技术将SIRT6-FLAG片段和质粒片段通过同源拼接得到表达质粒pET28TEV-SIRT6，其N端带His tag，C端带Flag tag。以pET28TEV-SIRT6为模板通过引物H133Y-F/R进行PCR定点突变，构建带有HIS tag、Flag tag双标签的SIRT6 H133Y的过表达质粒(pET28TEV-SIRT6H133Y)。
其中，SIRT6-FLAG片段的碱基序列(SEQ ID NO.1)为：
Figure PCTCN2022117402-appb-000005
质粒构建使用引物：
pET28a-F:gcggccgctttcgaatctagagc(SEQ ID NO.3)；
pET28a-R:gaattccggatccatggcgccctg(SEQ ID NO.4)；
SIRT6-F:gcgccatggatccggaattcatgagcgttaactatg(SEQ ID NO.5)；
SIRT6-R:ctagattcgaaagcggccgcttatttatcatcatcatc(SEQ ID NO.6)；
H133Y-F:cagaactgTATggtaatatgtttgttgaag(SEQ ID NO.7)；
H133Y-R:catattaccATAcagttctgccagtttatc(SEQ ID NO.8)。
(2)pET28TEV-SIRT6H133Y转化进大肠杆菌ArcticExpress(ED3)，转化子在2X YT培养基中(kanamycin 50μg/mL，gentamicin 40μg/mL)生长，OD 600达到0.8时，加入0.2mmol/L的IPTG，16℃过夜诱导蛋白表达。
(3)收集细菌菌液，8000rpm离心5min，弃上清，加入1mmol/L的PMSF裂解液(20mmol/L Tris-HCl，500mmol/L NaCl，2％glycerol，pH值为7.2)悬浮，用高压破碎仪进行细胞破碎， 12000rpm离心30min，取上清装入镍离子交换柱(GE Healthcare)，根据
Figure PCTCN2022117402-appb-000006
FPLC TM System进行纯化。加入10柱体积的wash buffer(20mmol/L Tris-HCl，500mmol/L NaCl，2％glycerol和20mmol/L Imidazole，pH值为7.2)洗涤，用elution buffer(20mmol/L Tris-HCl，500mmol/L NaCl，2％glycerol和500mmol/L imidazole，pH值为7.2)洗脱蛋白。SDS-PAGE电泳结果确认SIRT6 H133Y蛋白表达纯化成功(图2)。用透析buffer(50mmol/L Tris-HCl，250mmol/L NaCl and 10％glycerol，pH值为7.2)进行蛋白透析，去除咪唑，得到的蛋白保存于-80℃。
其中，SIRT6 H133Y蛋白的氨基酸序列(SEQ ID NO.2)(下换线突出为H133Y突变位点)：
Figure PCTCN2022117402-appb-000007
实施例2：
SIRT6敲低293T细胞株构建
用SIRT6shRNA病毒转染293T细胞，同时加入Polybrene增加感染效率，转染6h后换液(使用高糖DMEM培养基)。转染48h后用Puromycin进行阳性筛选，得到稳定表达SIRT6shRNA的细胞株。western blotting结果证明293T SIRT6敲低效率良好(图3)。
实施例3：
细胞培养及样品处理
(1)SIRT6敲低的293T细胞，用含有10％FBS的高糖DMEM培养基培养，待细胞汇合率达80％左右后，换低糖(1g/mL)的DMEM培养，其中加入Etomoxir(cpt1a抑制剂，中国aladdin公司)50μmol/L，1％的化学成分明确的脂质浓缩液，培养12h后收样，western blotting结果证明上述培养方法能够提高豆蔻酰化修饰丰度(图4)。细胞还可以通过培养基添加10μg/mL ALK14(豆蔻酸类似物)，培养12h后收样。
(2)细胞样品加入8mol/L的UA裂解液，冰上超声裂解(100W，工作10s，间歇10s，循环10次)，14000rpm离心30min后取上清得到蛋白裂解液测量浓度。取样品蛋白质20μg，加入5X上样缓冲液，沸水浴5min，进行12.5％SDS-PAGE电泳(120V，75min)，考马斯亮蓝染色证明蛋白质提取质量良好(图5)。取2mg蛋白样品加入10mmol/L的DTT在37℃孵育2h以破坏蛋白质二硫键，后冷却至室温，加入IAA至终浓度50mmol/L，避光孵育30min 用来修饰蛋白质SH基团。加入5倍体积水，将UA裂解液浓度稀释至1.5mol/L，按照50：1比例加入Trypsin，37℃酶切18h左右。通过SPE C18柱脱盐，冻干，得到酶解肽段，存放于-80℃备用。
实施例4：
豆蔻酰化修饰肽段富集
(1)酶解肽段(1mg)复溶于400μL预冷Myr-IP缓冲液中，随后加入80μL的SIRT6 H133Y蛋白(1mg/mL)和20μL乙腈，混匀，30℃反应4h，使SIRT6结合修饰肽段；
(2)取50μL的Anti-Flag磁珠于1.5mL的EP管中，加入500μL的Myr-IP缓冲液洗涤，混匀后将EP管放在磁力架上分离磁珠和液体，弃去液体，洗涤3次；
(3)将步骤(1)中的反应液与预处理好的Anti-Flag磁珠混匀进行富集反应，4℃孵育18h；
(4)将上述反应液从4℃取出，放在磁力架上分离，弃去液体；
(5)加入500μL的Myr-IP缓冲液洗涤，混匀后将EP管放在磁力架上分离磁珠和液体，弃去液体，重复洗涤3次；
(6)加入150μL洗脱缓冲液混匀，室温摇床孵育15min，放在磁力架上，将液体转入干净的1.5mL的EP管中，洗脱3次，混合；
(7)在洗脱产物中加入4％的TFA，37℃孵育10min后，12000rpm离心15min沉淀SIRT6蛋白，上清保留，可以直接作为质谱样品进行质谱鉴定或冷冻干燥保存。如果是冻干肽段用0.1％甲酸20％乙腈水溶液复溶作为质谱样品；
(8)真空干燥上清液，浓缩肽段。
Myr-IP缓冲液：1X PBS(不含Ca 2+、Mg 2+，pH 7.4)，0.2％Tween-20，20％乙腈。洗脱缓冲液：0.2mol/L甘氨酸，20％乙腈，pH值为2.2。
实施例5：
质谱鉴定
样品采用纳升流速的高效液相系统EASY-nLC 1200进行分离。流动相A为0.1％甲酸水溶液，流动相B为0.1％甲酸乙腈水溶液(乙腈为80％)。首先色谱柱以100％的流动相A进行平衡，随后样品的酶解肽段由自动进样器输送到上样柱(2cm，ID100μm，3μm，C18)，之后再经过分析柱(15cm，ID150μm，1.9μm，C18)进行分离，流速为600nL/min。肽段样品经分析色谱柱分离，HPLC肽段分离图说明肽段分离效果良好且丰度良好(图6)，后用Q Exactive HF-X质谱仪进行检测。检测方式为正离子模式，母离子扫描为700m/z，一级质谱分辨率为120,000at 200m/z，AGC(Automatic gain control)target为3e6，Maximum IT为30ms，动态排除时间(Dynamic exclusion)为12.0s。多肽和多肽碎片的质荷比按照下列方法采集：每次全扫描(full scan)后采集60个碎片图谱(MS2scan)，使用HCD碎裂模式，Normalized  Collision Energy为27％，Isolation window为1.6m/z，二级质谱分辨率7,500at 200m/z。
质谱分析采集的原始数据(RAW文件)，通过Thermo Proteome Discoverer软件进行数据库检索，最终获得蛋白质样品的鉴定信息。其中搜库参数设定如下：enzyme为Trypsin；missed cleavage sites设为2；非ALK14添加样品，动态修饰设定Oxidation(M)和Myr-(K)和Myr-(G)(质量迁移210.356Da)；ALK14添加样品，动态修饰设定Oxidation(M)和ALK14-(K)和ALK14-(G)(质量迁移234.377Da)。质谱数据分析后得到赖氨酸豆蔻酰化修饰靶蛋白及其修饰位点，DUX4L3和GLIS2为鉴定得到修饰蛋白中2个豆蔻酰化修饰蛋白(图7)。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中，而不必经过创造性的劳动。因此，本发明不限于上述实施例。本领域技术人员根据本发明的原理，不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。
Figure PCTCN2022117402-appb-000008
Figure PCTCN2022117402-appb-000009
Figure PCTCN2022117402-appb-000010
Figure PCTCN2022117402-appb-000011
Figure PCTCN2022117402-appb-000012
Figure PCTCN2022117402-appb-000013
Figure PCTCN2022117402-appb-000014

Claims (10)

  1. 一种SIRT6 H133Y蛋白的制备方法，其特征在于：其包括：
    以SIRT6-FLAG为模板通过SIRT6引物扩增得到SIRT6-FLAG片段，以pET28a质粒为模板通过pET28a引物扩增得到质粒片段，所述SIRT6-FLAG片段和质粒片段通过同源拼接得到表达质粒pET28TEV-SIRT6；以pET28TEV-SIRT6为模板，通过H133Y引物进行PCR定点突变技术，扩增得到过表达质粒pET28TEV-SIRT6H133Y；将所述过表达质粒pET28TEV-SIRT6H133Y转化大肠杆菌内，利用大肠杆菌蛋白质表达系统表达HIS tag、Flag tag双标签SIRT6H133Y蛋白，通过His标签纯化得到SIRT6 H133Y蛋白。
  2. 根据权利要求1所述的SIRT6 H133Y蛋白的制备方法，其特征在于：SIRT6-FLAG片段的碱基序列如SEQ ID NO.1所示；
    所述pET28a引物分别为如SEQ ID NO.3和SEQ ID NO.4所示；
    所述SIRT6引物分别如SEQ ID NO.5和SEQ ID NO.6所示。
  3. 根据权利要求1所述的SIRT6 H133Y蛋白的制备方法，其特征在于：所述得到过表达质粒pET28TEV-SIRT6H133Y时，PCR定点突变的扩增引物分别为如SEQ ID NO.7和SEQ ID NO.8所示；
    所述SIRT6 H133Y蛋白的氨基酸序列如SEQ ID NO.2所示。
  4. 一种SIRT6 H133Y蛋白，其特征在于：其由权利要求1-3任一项所述的制备方法得到。
  5. 一种利用权利要求4所述的SIRT6 H133Y蛋白进行SIRT6 H133Y蛋白富集豆蔻酰化修饰肽段的方法，其特征在于：其包括如下步骤：
    (1)、细胞样品或组织样品经过消化裂解提取全蛋白质，然后加入胰蛋白酶进行酶切，得到酶解肽段，冷干备用；
    (2)、将所述酶解肽段溶于缓冲液中，加入SIRT6 H133Y蛋白和乙腈混合，得到第一混合液；再将Anti-Flag磁珠加入缓冲液内，然后加入所述第一混合液进行富集反应，孵育，得到第二混合液；
    (3)、将所述第二混合液磁力沉淀，弃去液体，加入缓冲液洗涤，磁力沉淀，反复洗涤后，彻底弃去液体；
    (4)、洗涤后的Anti-Flag磁珠加入洗脱缓冲液，孵育，洗脱，得到肽段洗脱液；
    (5)、在所述肽段洗脱液中加入三氟乙酸进行孵育，离心，取上清液冻干，获得豆蔻酰化修饰肽段。
  6. 根据权利要求5所述的SIRT6 H133Y蛋白富集豆蔻酰化修饰肽段的方法，其特征在于：步骤(1)中，所述细胞样品，在DMEM低糖培养基内添加抑制剂和脂质、或在DMEM低 糖培养基内添加豆蔻酸类似物在37℃培养6-12h后得到；和/或，
    所述抑制剂为肉碱棕榈酰转移酶1a抑制剂，所述脂质为脂质浓缩液，所述豆蔻酸类似物为棕榈酸炔；和/或，
    步骤(1)中，所述组织样品包括人的各种组织和动物的各种组织。
  7. 根据权利要求5所述的SIRT6 H133Y蛋白富集豆蔻酰化修饰肽段的方法，其特征在于：
    步骤(1)中，所述酶切的温度为37℃，时间为16-18h。
  8. 根据权利要求5所述的SIRT6 H133Y蛋白富集豆蔻酰化修饰肽段的方法，其特征在于：步骤(2)和步骤(3)中，所述缓冲液为Myr-IP缓冲液，其包括磷酸缓冲盐溶液、吐温-20和乙腈；和/或，
    步骤(2)中，所述孵育的温度为4℃，时间为16-18h或室温2-4h；和/或，
    步骤(4)中，所述洗脱缓冲液包括甘氨酸和乙腈；和/或，
    步骤(5)中，所述孵育的温度为室温或者37℃，时间为5-10min；和/或，
    步骤(5)中，所述离心的转速为12000rpm，时间为15-30min。
  9. 豆蔻酰化修饰肽段，其特征在于：其由权利要求5-8任一项所述的方法得到。
  10. 一种如权利要求9所述的豆蔻酰化修饰肽段的应用，该豆蔻酰化修饰肽段在质谱鉴定中的应用。
PCT/CN2022/117402 2022-07-29 2022-09-06 Sirt6 h133y蛋白及其富集豆蔻酰化修饰肽段的方法和应用 WO2024021228A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210906268.6A CN115851686A (zh) 2022-07-29 2022-07-29 Sirt6 h133y蛋白及其富集豆蔻酰化修饰肽段的方法和应用
CN202210906268.6 2022-07-29

Publications (1)

Publication Number Publication Date
WO2024021228A1 true WO2024021228A1 (zh) 2024-02-01

Family

ID=85660438

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/117402 WO2024021228A1 (zh) 2022-07-29 2022-09-06 Sirt6 h133y蛋白及其富集豆蔻酰化修饰肽段的方法和应用

Country Status (2)

Country Link
CN (1) CN115851686A (zh)
WO (1) WO2024021228A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087619A (zh) * 2015-05-29 2015-11-25 苏州大学 一种类泛素修饰蛋白底物鉴定方法
CN105713957A (zh) * 2010-12-22 2016-06-29 康奈尔大学 Sirt6的调节物及其筛选分析
WO2021179214A1 (en) * 2020-03-11 2021-09-16 Shenzhen University Methods for enhancing anti-apoptotic pathway to prevent DOX toxicity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105713957A (zh) * 2010-12-22 2016-06-29 康奈尔大学 Sirt6的调节物及其筛选分析
CN105087619A (zh) * 2015-05-29 2015-11-25 苏州大学 一种类泛素修饰蛋白底物鉴定方法
WO2021179214A1 (en) * 2020-03-11 2021-09-16 Shenzhen University Methods for enhancing anti-apoptotic pathway to prevent DOX toxicity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GRIMLEY RACHEL, POLYAKOVA OXANA, VAMATHEVAN JESSICA, MCKENARY JOANNE, HAYES BRIAN, PATEL CHAMPA, SMITH JANET, BRIDGES ANGELA, FOSB: "Over Expression of Wild Type or a Catalytically Dead Mutant of SIRTUIN 6 Does Not Influence NFκB Responses", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 7, no. 7, US , pages e39847, XP093133664, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0039847 *
LISZT GREGORY, FORD ETHAN, KURTEV MARTIN, GUARENTE LEONARD: "Mouse Sir2 Homolog SIRT6 Is a Nuclear ADP-ribosyltransferase", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 280, no. 22, 1 June 2005 (2005-06-01), US , pages 21313 - 21320, XP093133668, ISSN: 0021-9258, DOI: 10.1074/jbc.M413296200 *
MARK A. KLEIN, CAN LIU, VYACHESLAV I. KUZNETSOV, JOHN B. FELTENBERGER, WEIPING TANG, JOHN M. DENU: "Mechanism of activation for the sirtuin 6 protein deacylase", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 295, no. 5, 31 January 2020 (2020-01-31), US , pages 1385 - 1399, XP055771341, ISSN: 0021-9258, DOI: 10.1074/jbc.RA119.011285 *

Also Published As

Publication number Publication date
CN115851686A (zh) 2023-03-28

Similar Documents

Publication Publication Date Title
Hwang et al. Expression of functional recombinant mussel adhesive protein type 3A in Escherichia coli
US20140287528A1 (en) Incorporation of methyl lysine into polypeptides
Lebert et al. Production and use of stable isotope-labeled proteins for absolute quantitative proteomics
Bunik et al. Novel isoenzyme of 2‐oxoglutarate dehydrogenase is identified in brain, but not in heart
White et al. UDP-N-acetyl-α-d-galactosamine: polypeptide N-acetylgalactosaminyltransferase: identification and separation of two distinct transferase activities
US20220299522A1 (en) Compositions and methods for identifying o-linked glycosylation sites in proteins
Synowsky et al. Probing Genuine Strong Interactions and Post-translational Modifications in the Heterogeneous Yeast Exosome Protein Complex* S
CN112904017B (zh) 一种基于共价连接的已知分子与蛋白质相互作用检测系统及其鉴定或验证方法
EP3722422B1 (en) Method for isolating dna by using cas protein system
JP6967002B2 (ja) トランスグルタミナーゼ認識部位を有するｆｋｂｐドメイン
Suzuki et al. Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system
Adelmant et al. Tandem affinity purification and mass spectrometry (TAP‐MS) for the analysis of protein complexes
Staes et al. Protease substrate profiling by N-terminal COFRADIC
CN109884163B (zh) 一种外膜蛋白酶t酶解组蛋白并进行质谱分析的方法
CN104610443B (zh) 一种高稳定性重组降钙素原、制备方法及用途
JP2022502070A (ja) プロテアーゼバリアントのスクリーニング方法および得られたプロテアーゼバリアント
CN109957003B (zh) 一种稳定的saa突变体及其在疾病检测中的应用
CN101581727A (zh) 一种高效检测体内蛋白相互作用的方法
WO2024021228A1 (zh) Sirt6 h133y蛋白及其富集豆蔻酰化修饰肽段的方法和应用
CN112574969A (zh) G6pdh突变体及其应用
Zhao et al. An efficient on‐column expressed protein ligation strategy: application to segmental triple labeling of human apolipoprotein E3
US20070134802A1 (en) Ionization modifier for mass spectrometry
Dangerfield et al. Enhancement of the StreptoTag method for isolation of endogenously expressed proteins with complex RNA binding targets
WO2021244001A1 (zh) 一种基于肽段的靶向蛋白质组精确定量方法
CN113666988A (zh) 新型冠状病毒核衣壳蛋白n端结构域的制备方法及应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22952669

Country of ref document: EP

Kind code of ref document: A1