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WO2017026890A1 - Procédé d'électrophorèse bidimensionnelle pour la séparation de composés protéiques - Google Patents

Procédé d'électrophorèse bidimensionnelle pour la séparation de composés protéiques Download PDF

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Publication number
WO2017026890A1
WO2017026890A1 PCT/MY2016/050030 MY2016050030W WO2017026890A1 WO 2017026890 A1 WO2017026890 A1 WO 2017026890A1 MY 2016050030 W MY2016050030 W MY 2016050030W WO 2017026890 A1 WO2017026890 A1 WO 2017026890A1
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Prior art keywords
fractions
separation
liquid phase
eluted
dimension
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Ceased
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PCT/MY2016/050030
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English (en)
Inventor
Mohd Nazri BIN ISMAIL
Soon Hong Kwan
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Universiti Sains Malaysia (USM)
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Universiti Sains Malaysia (USM)
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Publication of WO2017026890A1 publication Critical patent/WO2017026890A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—Electrophoresis
    • 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
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/24—Extraction; Separation; Purification by electrochemical means
    • C07K1/26—Electrophoresis
    • C07K1/28—Isoelectric focusing
    • 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
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

Definitions

  • This invention relates to the field of electrophoretic procedures, more particularly to an inverted two-dimensional electrophoretic method which adopts gel eluted liquid fraction entrapment electrophoresis (Gelfree) technique in the first dimension followed by isoelectric focusing in the second dimension.
  • Gelfree gel eluted liquid fraction entrapment electrophoresis
  • Electrophoresis is used to separate complex substances into their component parts by using procedures based upon the migration of electrically charged fractions in a direct current electric field.
  • this technique has been done using a one dimensional (ID) system in a support medium such as a polyacrylamide gel (PAGE) with the addition of denaturing agents, such as sodium dodecyl sulfate (SDS) or urea, which provides a separation based on mass or on a mass-to-charge ratio.
  • Two dimensional (2D) electrophoresis system begins with ID electrophoresis but then separates the molecules by a second property in a direction 90 degrees from the first.
  • 2D electrophoresis is a technique used to separate protein compounds based on isoelectric points in the first dimension, followed by another separation based on molecular weight of each separated compound in the second dimension, in which sodium dodecyl- sulfate (SDS) and buffer are incorporated into the polyacrylamide gel in the latter.
  • SDS sodium dodecyl- sulfate
  • Such technique was introduced in article by Patrick H. O' Farrell in The Journal of Biological Chemistry Vol. 250, No. 10, Issue of May 25, pp. 4007-4021, 1975. Such method was also exemplified in United States patent no.
  • Tuszynski et al. disclosed a 2D-PAGE system using SDS-PAGE in the first dimension and polyacrylamide gel isoelectric focusing (PAGIF) in the second dimension in Analytical Biochemistry 93, 329-338 (1979).
  • Tuszynski et al. disclosed an additional step of subjecting gradient gel used in the first dimension to another electrophoresis in 4.5 % acrylamide-SDS stacking gel such that the proteins in the first dimension could easily migrate into the PAGIF gel in the second dimension based on a steady- state stacking principle.
  • proteins are allowed to dissociate bound SDS and focus in accordance with intrinsic charge properties in the second dimension.
  • this invention provides an inverted 2D-PAGE method which adopts gel eluted liquid fraction entrapment electrophoresis (Gelfree) technique in the first dimension followed by isoelectric focusing in the second dimension to eliminate the use of SDS from 2D-PAGE.
  • This invention provides a two-dimensional inverted 2D-PAGE method which provides comparative result to a two-dimensional SDS-PAGE method, yet eliminating the need to use SDS in the method.
  • This invention provides determination of molecular weights and approximate isoelectric points of proteins.
  • This invention provides a convenient method for analysis and detection of proteins from complex biological sources, such as ribosomal assembly, plant extract, bacteria and/or highly insoluble proteins such as periphilin.
  • This invention is capable of resolving proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge.
  • one of the embodiments of the present invention is a method of separating protein compounds from a sample based on inverted two-dimensional polyacrylamide gel electrophoresis (i2D-PAGE) comprising the steps of: electrophoretically separating compounds from a sample loaded into a polyacrylamide gel tube in a first dimension into discrete fractions of different molecular weights, in which the separation is conducted using gel eluted liquid fraction entrapment electrophoresis whereby each of the fractions being eluted from the gel tube is trapped in a subsequent liquid phase; collecting the liquid phase containing each of the eluted fractions; and subjecting the liquid phase to a second separation in a second dimension using an isoelectric focusing medium in the presence of electric field, in which each of the fractions are resolved according to its isoelectric point.
  • i2D-PAGE inverted two-dimensional polyacrylamide gel electrophoresis
  • the method further comprises a step of treating the sample in the presence of reducing agent selected from the group consisting of dithiothreitol and 2- mercaptoethanol.
  • the method further comprises a step of solubilizing the eluted fractions in each of the collected liquid phase in the presence of nonionic surfactant selected from any one or a combination of polysorbate 20, 40 and 80.
  • the method further comprises a step of reducing volume of the collected liquid phase to an amount suitable for the isoelectric focusing medium used.
  • the method further comprises a step of staining the isoelectric focusing medium with Coomasie blue or silver stain.
  • the isoelectric focusing medium is treated with trichloroacetic acid prior to the staining step.
  • the gel eluted liquid fraction entrapment electrophoresis in conducted in the presence of voltage from 50 to 100 V.
  • the second separation is conducted in the presence of electric field provided at electrical settings at voltage of 2000 V, current of 50 mA, power of 30 W.
  • the isoelectric focusing medium used in the separation in the second dimension comprises urea, sucrose, water, ampholytes, acrylamide, bisacrylamide, tetramethylethylenediamine and ammonium persulfate.
  • one of the embodiments of the present invention is a method of separating protein compounds from Sargassum polycystum plant extract based on inverted two-dimensional polyacrylamide gel electrophoresis (i2D-PAGE) comprising the steps of: electrophoretically separating compounds from a sample loaded into a polyacrylamide gel tube in a first dimension into discrete fractions of different molecular weights, in which the separation is conducted using gel eluted liquid fraction entrapment electrophoresis whereby each of the fractions being eluted from the gel tube is trapped in a subsequent liquid phase; collecting the liquid phase containing each of the eluted fractions; and subjecting the liquid phase to a second separation in a second dimension using an isoelectric focusing medium in the presence of electric field, in which each of the fractions are resolved according to its isoelectric point.
  • i2D-PAGE inverted two-dimensional polyacrylamide gel electrophoresis
  • Fig. 1 shows separation result of Sargassum polycystum extract using method disclosed in the preferred embodiment.
  • the present invention relates to an inverted 2D-PAGE (i2D-PAGE) method which adopts gel eluted liquid fraction entrapment electrophoresis (GelFrEE) technique in the first dimension followed by isoelectric focusing in the second dimension.
  • i2D-PAGE inverted 2D-PAGE
  • GelFrEE gel eluted liquid fraction entrapment electrophoresis
  • the present invention disclosed a method of separating protein compounds from a plant extract sample.
  • a plant extract sample For instance, a Sargassum polycystum extract, but not limited thereto.
  • parameters used for separation of protein compounds may vary according to different sample.
  • the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.
  • the method of separating protein compounds from the Sargassum polycystum extract based on inverted two-dimensional polyacrylamide gel electrophoresis comprising the steps of: electrophoretically separating compounds from a sample loaded into a polyacrylamide gel tube in a first dimension into discrete fractions of different molecular weights, in which the separation is conducted using gel eluted liquid fraction entrapment electrophoresis whereby each of the fractions being eluted from the gel tube is trapped in a subsequent liquid phase; collecting the liquid phase containing each of the eluted fractions; and subjecting the liquid phase to a second separation in a second dimension using an isoelectric focusing medium in the presence of electric current, in which each of the fractions are resolved according to its isoelectric point.
  • i2D-PAGE inverted two-dimensional polyacrylamide gel electrophoresis
  • the sample Prior to separation of the extract, it is preferred that the sample is suitably treated for satisfactory separation thereinafter.
  • the extract be prepared by mixing with a sample buffer, a reducing agent and water.
  • the sample buffer can be acetate buffer but not limited thereto.
  • the reducing agent can be dithiothreitol (DTT) whereas the water used is preferably purified water.
  • the sample to be loaded into the polyacrylamide gel tube in the first dimension comprises up to 112 of the extract, 30 ⁇ L of the sample buffer, 8 ⁇ L of 1M DTT and, if necessary, certain volume purified water to bring the final sample volume to 150 L.
  • the prepared sample is preferably loaded into a fractionation system capable of performing gel eluted liquid fraction entrapment electrophoresis (Gelfree) thereby separating compounds in the sample into discrete fractions of different molecular weights in the presence of 50 to 100 V. More preferably, the prepared sample is loaded into a fractionation system capable of providing liquid-phase molecular weight fractions hence enabling liquid-phase recovery of the eluted fractions. More preferably, the sample is loaded into a system designed for separation in the mass range of 3.5 to 150 kDa, with resolution between 15 kDa and 100 kDa.
  • the preferred system essentially comprises a complementary fractionation cartridge having polyacrylamide gel tube, power supply capable of providing constant voltage sufficient for the separation, electrode arrays and computer with user programming to control and monitor the system.
  • the polyacrylamide gel tube in the cartridge comprises a gel matrix which comprises organic amine and an anionic compound selected from the group consisting of, preferably, acetate, thereby being generally known as Tris-acetate.
  • one exemplary fractionation system can be found under the trade name of Gelfree 8100 Fractionation System while its complementary catridge can be found under the trade name of Gelfree 8100 catridge kit 10 % Tris-acetate.
  • anionic buffer reservoir, cationic buffer reservoir and sample collection chamber in the catridge comprises 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid (HEPES) buffer in different amount.
  • HEPES 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid
  • the user programming is preset to pause at pre-defined pause intervals for collection of each fraction eluted into the liquid phase in the sample collection chamber. More preferably, the separation by gel eluted liquid fraction entrapment electrophoresis herein is paused at intervals of 2 to 36 mins. In the preferred embodiment, the Sargassum poly cy stum extract is separated into 12 fractions after 160 mins in the first dimension separation.
  • the method further comprises a step of solubilizing the eluted fractions in each of the collected liquid phase in the presence of nonionic surfactant selected from any one or a combination of polysorbate 20, 40 and 80.
  • the step of solubilizing the eluted fractions enables protein to be soluble therein so as to give good separation in the next dimension.
  • the step of solubilizing the eluted fractions can be conducted in the presence of heat at a temperature sufficiently dissolving the protein but not destroying it.
  • the step of solubilizing the eluted fractions is conducted at a temperature of 75 to 85 °C, more preferably at 80 °C.
  • the method further comprises a step of reducing volume of the collected liquid phase to a volume which is suitable for the next separation step in the second dimension.
  • the volume suitable for the next separation step depends on the capacity of isoelectric focusing wells of the isoelectric focusing gel.
  • each of the collected liquid phase is preferably reduced to a volume of 25 ⁇ L.
  • a vacuum concentrator or the like can be employed to conduct this step.
  • the collected fractions are preferably separated using isoelectric focusing (IEF) technique in the second dimension.
  • IEF isoelectric focusing
  • the collected fractions are further separated based on differences in their isoelectric point (pi).
  • IEF technique is conducted based on principle known in the art.
  • the method employs an IEF gel as IEF medium where the eluted fractions from GelFrEE are disposed thereto.
  • the gel comprises ampholyte to establish pH gradient.
  • the isoelectric focusing (IEF) gel used herein is preferably horizontally casted to resemble a flatbed gel electrophoresis system.
  • the IEF gel herein preferably comprises urea, sucrose, purified water, ampholytes, acrylamide, bisacrylamide, trimethylethylenediamine (TEMED), and ammonium persulfate (APS) to establishment of pH gradient therein when electric field is applied.
  • the step of separation in the second dimension employs a cathode buffer comprises ampholytes pH 6-8 solution and purified water.
  • an anode buffer comprises orthophosphoric acid and purified water is preferred.
  • the IEF gel comprises 13.3 g of urea, 1.55 g of sucrose, 12.4 mL of purified water, 3.4 mL of ampholytes, 5.5 mL of acrylamide/bisacrylamide, 31 ⁇ , of TEMED and 310 ⁇ ., of APS.
  • the ampholyte used is a carrier ampholyte 4-6 thereby enable establishment of pH gradient in a range of 4 to 6.
  • the second separation in the second dimension is preferably conducted in the presence of electrical field provided at electrical settings at voltage of 2000 V, current of 50 mA, power of 30 W for a duration of 1 to 2 hours. More particularly, the second separation is preferably conducted in the presence of electrical settings at voltage of 2000 V, current of 50 mA, power of 30 W for a duration of 3.5 hours.
  • the method further comprises a step of staining the isoelectric focusing medium with Coomasie blue or silver. More preferably, Coomasie blue.
  • the isoelectric focusing medium is treated with trichloroacetic acid prior to the staining step.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Electrostatic Separation (AREA)

Abstract

La présente invention concerne un procédé de séparation de composés protéiques d'un échantillon basé sur une électrophorèse sur gel de polyacrylamide bidimensionnelle inversée (i2D-PAGE) comprenant les étapes consistant à : séparer par électrophorèse des composés d'un échantillon chargé dans un tube de gel de polyacrylamide dans une première dimension en fractions distinctes de masses moléculaires différentes, ladite séparation étant effectuée à l'aide d'une électrophorèse avec piégeage des fractions liquides éluées sur le gel, moyennant quoi chacune des fractions éluées du tube de gel est piégée dans une phase liquide ultérieure ; recueillir la phase liquide contenant chacune des fractions éluées ; et soumettre la phase liquide à une seconde séparation dans une seconde dimension à l'aide d'un milieu de focalisation isoélectrique en présence d'un champ électrique, chacune des fractions étant résolue selon son point isoélectrique.
PCT/MY2016/050030 2015-08-13 2016-05-06 Procédé d'électrophorèse bidimensionnelle pour la séparation de composés protéiques Ceased WO2017026890A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
MYPI2015702656A MY190479A (en) 2015-08-13 2015-08-13 A two-dimensional electrophoresis method for separation of protein compounds
MYPI2015702656 2015-08-13

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WO2017026890A1 true WO2017026890A1 (fr) 2017-02-16

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105527332A (zh) * 2016-01-18 2016-04-27 云南省农业科学院花卉研究所 百合总蛋白的提取及双向电泳差异蛋白质图谱的获取方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100270159A1 (en) * 2007-10-09 2010-10-28 Doucette Alan A Apparatus for Purifying Molecules

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100270159A1 (en) * 2007-10-09 2010-10-28 Doucette Alan A Apparatus for Purifying Molecules

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KIM ET AL.: "Online Matrix Removal Platform for Coupling Gel-Based Separations to Whole Protein Electrospray Ionization Mass Spectrometry", JOURNAL OF PROTEOME RESEARCH, vol. 14, no. 5, 1 May 2015 (2015-05-01), pages 2199 - 2206, XP055364677 *
SKINNER ET AL.: "Native GELFrEE: A New Separation Technique for Biomolecular Assemblies", ANALYTICAL CHEMISTRY, vol. 87, no. 5, 3 March 2015 (2015-03-03), pages 3032 - 3038, XP055364670 *
TRAN ET AL.: "Gel-Eluted Liquid Fraction Entrapment Electrophoresis: An Electrophoretic Method for Broad Molecular Weight Range Proteome Separation", ANALYTICAL CHEMISTRY, vol. 80, 2008, pages 1568 - 1573, XP055364692 *
TRAN ET AL.: "Multiplexed Size Separation of Intact Proteins in Solution Phase for Mass Spectrometry", ANALYTICAL CHEMISTRY, vol. 81, 2009, pages 6201 - 6209, XP055364653 *
TUSZYNSKI ET AL.: "A Two-Dimensional Polyacrylamide Gel Electrophoresis (PAGE) System Using Sodium Dodecyl Sulfate-PAGE in the First Dimension", ANALYTICAL BIOCHEMISTRY, vol. 93, 1979, pages 329 - 338 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105527332A (zh) * 2016-01-18 2016-04-27 云南省农业科学院花卉研究所 百合总蛋白的提取及双向电泳差异蛋白质图谱的获取方法
CN105527332B (zh) * 2016-01-18 2018-05-22 云南省农业科学院花卉研究所 百合总蛋白的提取及双向电泳差异蛋白质图谱的获取方法

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