WO2012159769A1 - Electrophorèse par gel 2d améliorée - Google Patents

Electrophorèse par gel 2d améliorée Download PDF

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Publication number
WO2012159769A1
WO2012159769A1 PCT/EP2012/002252 EP2012002252W WO2012159769A1 WO 2012159769 A1 WO2012159769 A1 WO 2012159769A1 EP 2012002252 W EP2012002252 W EP 2012002252W WO 2012159769 A1 WO2012159769 A1 WO 2012159769A1
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Prior art keywords
gel
proteinaceous
protein
sample
standard
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PCT/EP2012/002252
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English (en)
Inventor
Simone Koenig
Doreen ACKERMANN
Wang WEIQUN
Lothar GRUEN
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Universitaetsklinikum Muenster
Eco-Luftqualitaet + Raumklima Messstelle, Beratungs- Und Forschungsgesellschaft Mbh
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Priority to EP12729346.2A priority Critical patent/EP2715331A1/fr
Publication of WO2012159769A1 publication Critical patent/WO2012159769A1/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
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
    • 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
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • 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
    • G01N27/44743—Introducing samples
    • 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
    • G01N27/44747—Composition of gel or of carrier mixture
    • 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
    • G01N27/44782—Apparatus specially adapted therefor of a plurality of samples
    • 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
    • G01N27/44795—Isoelectric focusing

Definitions

  • the present invention relates to a method for separating a plurality of proteins which method comprises providing a labeled proteinaceous sample or more than one proteinaceous samples which are distinguishably labelled; providing a proteinaceous standard which is distinguishable from said label of said proteinaceous sample(s); subjecting said proteinaceous sample(s) to isoelectric focusing in a pH gradient gel; subjecting the standard to stacked gel electrophoresis; subjecting said proteinaceous sample(s) and said standard to resolving gel electrophoresis; and comparing the separated proteinaceous entities and the separated internal standard.
  • the inventions methods may also be used for identifying3 ⁇ 4the presence of one or more proteins contained in a proteinaceous sample, or for fietermining the allergenic, carcinogenic, contamination or infection potential of a proteinaceous sample.
  • a gel suitable for gel electrophoresis of proteins comprising (from top to down) a stacking gel; a predetermined pH gradient gel; and a resolving gel is also disclosed.
  • the methods may also be used for identifying the quality of the gel matrix, i.e3 ⁇ 43 ⁇ 4s a quality control. If so, the marker is preferably separated without a mixture to be
  • Gel electrophoresis is a basic analytical technique which is widely used in most biochemical/analytical laboratories in academia and industry. Gel electrophoresis is mainly used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric field applied to a gel matrix.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • protein molecules using an electric field applied to a gel matrix.
  • a special kind of gel electrophoresis is the so-called “2D gel electrophoresis", “two-dimensional polyacrylamide gel electrophoresis” or simply “2D-PAGE” (or 2D, sometimes also used herein is “2-DE”), in which proteins are separated according to charge (isoelectric point pi) by isoelectric focusing (IEF) in the first dimension and according to size (molecular weight MW) by sodium dodecyl sulfate (SDS)-PAGE in the second dimension.
  • This technique has a unique capacity for the resolution of complex mixtures of proteins, permitting the simultaneous analysis of hundreds or even thousands of gene products (see Figure 1).
  • 2D electrophoresis usually begins with isoelectric focusing which makes use of one property of the target protein, i.e., the isoelectric point allows the separation of proteins or other molecules in accordance with their isoelectric point.
  • Isoelectric focusing takes place in a special shaped apparatus. Since the proteins or other molecules in question are separated in one dimension only, all the proteins will be aligned in a single strip. These proteins are then separated in a further special shaped apparatus by way of a second property (normally the protein mass/molecular weight) in a direction 90 degrees from the first. Because it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated in 2D electrophoresis than in 1D electrophoresis.
  • isoelectric focusing a pH-gradient is applied to a gel strip and an electric potential is applied across the gel, making one end of the resulting gel more positive than the other.
  • proteins will be charged. If they are positively charged, they will be pulled towards the negative end of the pH-gradient within the gel and if they are negatively charged they will be pulled to the positive end of the gel.
  • the proteins applied in the first dimension will therefore travel along the pH-gradient within the gel and will accumulate at their isoelectric point - said isoelectric point is the point at which the overall charge of the protein is about 0 (neutral).
  • the proteins are separated by their mass.
  • they are treated with a charged carrier like sodium dodecyl sulfate (SDS - a negatively charged carrier).
  • SDS sodium dodecyl sulfate
  • This treatment denatures the proteins and at the same time binds a number of carrier molecules (for example SDS) roughly proportional to the protein's length.
  • carrier molecules for example SDS
  • SDS charged carrier molecules
  • the negatively charged (due to the SDS) proteins will travel to the more positive side of the gel proportionally to their mass-to-charge ratio. Since the gel acts like a molecular sieve when the current is applied, the proteins are separated on the basis of their molecular weight.
  • DIGE differential fluorescence gel electrophoresis
  • DIGE is a commercially available, well-known technique which was developed by Amersham/GE Healthcare. DIGE is characterized in that similar proteomes (e.g. stimulated versus normal cells) are compared on one single gel by the use of three fluorescent dyes which can be separated by a special laser scanning technique. The description of DIGE by the manufacturer is as follows (see also [2]): 2D Fluorescence Difference Gel Electrophoresis (2D DIGE) is a method that labels protein samples with fluorescent dyes before 2D electrophoresis, enabling accurate analysis of differences in protein abundance between samples. It is possible to separate up to three different samples within the same 2D gel.
  • the internal standard is prepared by mixing together equal amounts of each sample in the experiment and including this mixture on every gel within an experimental series. Normalization of the internal standard across gels allows the ratio of relative abundance of the same protein in different samples to be compared directly. Even small differences in expression levels can be determined by comparing the ratio obtained from one fluorescently labeled sample directly with another.
  • DIGE represents a further step on the way down to a suitable, reliable method allowing the analysis of very complex proteomes
  • DIGE like a conventional 2D-gel electrophoresis, still encounters problems.
  • DIGE gels (and the "conventional" 2D-gels as such) may not always allow a reliable and safe allocation of protein spots which are produced by one and the same protein on different gels. This is so, because the actual "running" behavior of a protein differs significantly when it is analyzed on different 2D-gels. It may in fact be extremely difficult to allocate one and the same protein signal on different 2D-gels.
  • DIGE is not capable to achieve the allocation of spots of samples containing proteins that may be quite different from each other.
  • DIGE is useful for comparing quite similar or only slightly different proteins contained in a sample, such as the proteome of a healthy and cancerous cell of the same origin (e.g., breast, bladder, kidney) or the proteome of an induced or non-induced cell.
  • DIGE allows the direct comparison of samples on the same gel, however, this comparison is limited to the abundance of proteins.
  • a common technique in DIGE is to include an internal standard in each gel. The internal standard is prepared by mixing together several or all of the samples in the experiment. This allows the measurement of the abundance of a protein in each sample relative to the internal standard. Since the amounts of each protein in the internal standard is known to be the same in every gel, this method reduces inter-gel variation.
  • DIGE or any other 2D-technique reaches its limits when two different proteinaceous samples should be compared or a "new" proteinaceous sample should be compared with an "old" one, for example, a new probe should be compared with a proteinaceous sample run a couple of years ago.
  • a method nor a technique is available which allows the standardized allocation of spots of a 2D-gel, if two or more proteinaceous samples are run on the same gel or, if two or more gels should be compared with each other.
  • the present invention addresses this need and provides means and methods which will allow the safe and reliable allocation of protein spots on the same or on different 2D-gels.
  • 2Delectrophoresis is a two-step procedure using two different devices. Typically, 2D electrophoresis begins with isoelectric focussing and then separates the molecules by a second property in a direction 90 degrees from the first. In 1D electrophoresis, proteins (or other molecules) are separated in one dimension, so that all the proteins/molecules will lie along a lane, while the molecules are spread out in a 2 nd dimension across a 2D gel. Because it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated in 2D electrophoresis than in 1D electrophoresis. For orientation a marker mixture can be applied.
  • the proteins separated via 2D electrophoresis can then be detected by a variety of means, but the most commonly used stains are silver and Coomassie Brilliant Blue staining.
  • the most difficult task is to identify the protein spots that are of interest. Both software and specialized picking roboters are available which match spots between gels of similar samples to show, for example, proteomic differences between early and advanced stages of an illness. While this technology is widely utilized, the intelligence has not been perfected. For example, while two algorithms tend to agree on the quantification and analysis of well-defined well- separated protein spots, they may deliver different results and analysis tendencies with less-defined less-separated spots. To this end, challenges in allocation a protein spot to a distinct protein include:
  • DIGE differential fluorescence gel electrophoresis
  • DIGE or any other comparable technique has further limitations, if the protein source, quantity and chemical background of a mixture of molecules, in particular a mixture of proteins that should be compared, varies. Furthermore, DIGE or any other comparable technique is not suitable to be applied in a scenario where a sample molecule profile, in particular a sample protein profile assembled in a database today remains comparable to those profiles added in the future.
  • scientists have been battling the analytical problem of gel matching for years. In practice, mostly only the visual comparison or a rough overlay of 2D-gels is possible, not an exact protein spot matching, in particular, if gels have been generated in different laboratories around the world.
  • the present inventors envisioned the use of marker proteins labelled with one fluorescent dye while the individual sample was labelled with the other, thereby providing a grid that provides, so to say, the possibility to determine coordinates in reference to the marker gel image.
  • the present inventors created a regular grid of protein spots across a 2D-gel (so called "Grid Gels") with the goal of reproducibly having at least 3 x 3, but better n x m (n, m >5) nodes.
  • the methodology developed by the present inventors thus allows the in/on-gel generation of a reference protein spot grid that thus provides reference spots which can be used to determine/compare the position of an unknown protein spot (of interest).
  • the position of the reference spot can be set in relation to the position of an unknown spot (of interest), thereby allowing the generation of relative values to allocate a certain position (or coordinates) to said unknown spot (of interest).
  • proteinaceous samples e.g., clinical, environment or alimentary sample
  • a reference profile from, e.g., clinics, environmental or food sources
  • the grid generated by the internal standard remains (or will be) the same such that based on known relative values (i.e., relation between the position of the reference spot and position of a protein spot of interest) a protein spot of interest can be determined.
  • the means and methods of the present invention allow that sample protein profiles assembled in a database should remain comparable to those added in the future.
  • the present invention provides a comparative fluorescence gel electrophoresis by using a combination of a gel-strip sandwich (GSS) and polyacrylamide gel electrophoresis (PAGE) which is called CoFGE.
  • GSS gel-strip sandwich
  • PAGE polyacrylamide gel electrophoresis
  • the present invention relates in one aspect to a method for separating a plurality of proteins which method comprises:
  • the present invention relates in a further preferred embodiment to a method for separating a plurality of proteins which method comprises:
  • the stacking gel forms the upper part of the gel where the standard is applied, followed by the pH gradient gel in which the proteinaceous sample(s) have been made subject to isoelectric focusing, followed by the resolving gel in which both, standard and proteinaceous sample(s), are made subject of resolving gel electrophoresis. It is also preferred that the stacking gel comprises at least one or more, i.e. two, three, four, five, six, seven, eight, nine, ten, or even more gel pockets in parallel.
  • the stacking gel comprises gel pockets in the stacking gel that are arranged such that they span/cover (relating to the long side of the pH gradient gel, which long side is parallel to the running direction of the pH gradient gel) preferably that part of the pH gradient gel which comprises the proteinaceous sample(s) which already have been made subject to isoelectric focusing in a pH gradient gel.
  • the methods of the invention can be characterized by a method comprising: (a) optionally providing a labeled proteinaceous sample or more than one proteinaceous samples which samples are distinguishably labelled;
  • the pH gradient gel is prepared such that it contains slots along one of its long sides (wherein the long side is preferably parallel or nearly parallel to the running direction of the pH gradient gel) and that the proteinaceous standard(s) are loaded onto/into these slots. It follows that it is also envisaged to either minimize the stacking gel or even leave it out, provided that the pH gradient contains slots (for example by way of punching them out) and is able to stack the proteinaceous standard(s). Also this assembly may be used in the context of all embodiments of the present invention.
  • the step of subjecting the standard to stacked gel electrophoresis is preferably conducted such that a grid of defined proteinaceous spots (resulting from the proteinaceous standard(s)) is obtained on the resolving gel. It follows that at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more slots of a stacking gel are loaded with (preferably one and the same) standard and are subject to stacked gel electrophoresis.
  • the stacking gel can be replaced by the pH gradient gel, provided that the latter contains slots and provided that the latter pH gradient gel is composed such that it is able to stack the proteinaceous standard. Stacking gel and pH gradient gel are thereby one and the same gel.
  • the pH gradient gel is run first and, thereafter, the proteinaceous standard(s) is(are) applied in the slots that have to be arranged on the long side (parallel to the running direction of the pH gradient gel during the isoelectric focusing). These slots may be punched out.
  • method (b) comprises a step (f) comparing the separated proteinaceous entities and the separated internal standard and thereby identifying the presence of one or more proteins contained in a proteinaceous sample; and method (c) comprises a step (f) comparing the separated proteinaceous entities and the separated internal standard and thereby determining the allergenic, carcinogenic, contamination or infection potential of said proteinaceous sample.
  • the comparison step allows the determination of spot coordinates, since the internal standard generates the "grid" to which protein spots could be set in relation, meaning that coordinates for a spot (from a molecule, in particular a protein) can be determined by reference to (or in relation to) the internal standard.
  • the methods of the present invention allow the generation of an artificial protein grid on the same gel on which a sample to be analyzed was run. Different fluorescent stains are used to distinguish samples on the gel.
  • the technology applied in the methods described herein combine elements of 1D and 2D.
  • xD or “xDE” (with x being 1 or 2) means 1 dimensional or 2 dimensional (gel) electrophoresis, respectively.
  • x being 1 or 2
  • xDE gel electrophoresis
  • Dependent on the context 1D or 2D electrophoresis also includes 1 D or 2D polyacrylamide (gel) electrophoresis.
  • the skilled artisan is aware of the context-dependent meaning because of his/her common general knowledge.
  • special gel combs with V-shaped wells are provided which are placed above the pi strip. Proteins separated on the pi strip are electrophoresed at the same time as marker proteins placed in preferably V-wells. In that way, grids providing preferably -100 nodes as landmarks for the determination of protein spot coordinates are then generated. Data analysis is possible with regular 2D software and may optionally require manual control.
  • the anchor (or landmark) reference spots also referred to herein as "reference spots” allow the allocation/assignment of protein spot coordinates, i.e., of protein spots of interest.
  • the position of a protein spot of interest can be set in relation to the position of a reference spot in order to determine its location, for example, by way of a relative value (position of the reference spot in relation to a protein spot of interest).
  • the method of the present invention improves comparability of 2D- gels, since they are hooked together with in-gel anchor points during an experiment (see Figure 1). Accordingly, in various preferred embodiments gel matching is achieved as long as the same size of pl-strips and protein markers are used.
  • separating means that a plurality of proteins such as a plurality of proteins comprised, for example, in a proteinaceous sample are separated because of their molecular weight and/or isoelectric point.
  • the separation is preferably achieved by gel electrophoresis such as 2D gel electrophoresis or DIGE with the modification achieved by the present invention as described herein.
  • the preferred gel electrophoresis is PAGE including native PAGE or SDS-PAGE.
  • a polyacrylamide gel is a separation matrix used in electrophoresis of biomolecules, such as proteins.
  • Polyacrylamide gels separate biomolecules differing by their molecular weight. It is currently most often used in the field of protein analysis, e.g. to separate different proteins or isoforms of the same protein into separate bands or spots.
  • a polyacrylamide gel has a stacking and resolving gel.
  • resolving gels are made using, for example, 6%, 7%, 8%, 9%, 10%, 11%, 12% or 15% acrylamide .
  • Stacking gel (such as 2%, 3%, 4%, 5% or 7%) is poured on top of the resolving gel and a gel comb (which forms the wells and defines the lanes where proteins, sample buffer and ladders will be placed) is inserted.
  • the percentage chosen depends on the size of the protein that one wishes to identify or probe in the proteinaceous sample. It is a general rule that the smaller the known weight, the higher the percentage that should be used.
  • the stacking gel is "colored" with bromphenolblue (BPB).
  • BBP bromphenolblue
  • This coloring facilitates visibility of the front, i.e., the front of the mixture, preferably proteinaceous mixture during its separation. Besides, the coloring facilitates loading of the slots with the mixture to be separated.
  • stacked gel electrophoresis can be vertical or horizontal stacked gel electrophoresis, with vertical being preferred.
  • resolving gel electrophoresis can be vertical or horizontal resolving gel electrophoresis. Accordingly, in some preferred embodiments both stacked gel electrophoresis and resolving gel electrophoresis may be vertical or horizontal. Likewise, stacked gel electrophoresis may be vertical and resolving gel electrophoresis may be horizontal and vice versa.
  • a “proteinaceous sample” is a sample comprising, inter alia, a plurality of proteins. Accordingly, said sample can comprise compounds different from proteins such as carbohydrates and lipids.
  • the sample may be of clinical, environmental or alimentary source.
  • the sample may comprise proteins from cells and/or organelles.
  • the term "cell” or “cells” as used herein is used broadly and is meant to cover eukaryotic and prokaryotic cells, including for example plant cells, mammalian cells, parasites, unicellular organisms, yeasts, fungi and bacterial cells.
  • organelle which is well-known in the art is meant to cover any cellular organelle from cells. Non- limiting examples thereof include vacuoles, mitochondria and chloroplasts.
  • more than one proteinaceous sample includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more proteinaceous samples.
  • a "plurality" of proteins may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, 300, 400, 500, 600, 700, 800, 900, 1000 or more proteins.
  • a “proteinaceous standard” is a mixture of proteins (including glycoproteins, phosphoproteins, or other modified proteins), wherein the proteins of the mixture differ in molecular weight and thus in their size.
  • proteins include glycoproteins, phosphoproteins, or other modified proteins
  • PAGE polyacrylamide electrophoresis
  • the proteinaceous standard preferably generates a grid on the gel, i.e., the standard generates "landmarks" which can be used as orientation points in order to allow the allocation to a distinct protein. This is the main difference to prior art methods such as 2D gel electrophoresis or DIGE.
  • the standard is subject to stacked gel electrophoresis in that at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more slots of a stacking gel are loaded with one and the same standard and are subject to stacked gel electrophoresis. It is also envisaged to punch the slots into the stacking gel. It is either envisaged that all slots of the stacking gel are loaded with the same standard, or just every other slot (i.e. one slot empty one loaded), or every third, or every forth slot, etc. It is envisaged that the standard is preferably evenly distributed in as many slots as necessary depending on the circumstances.
  • each and every slot comprises one and the same standard but that different standards are used, for example two different standards which are used for example alternating etc. (slot 1 - standard 1 ; slot 2 standard 2, slot 3 standard 1 , slot 4 standard 2 etc to give just one example.).
  • the skilled person is well aware how to choose and distribute the standard(s) in order to arrive at a reliable grid providing enough information (coordinates) which allows for example the comparison between different gels or different samples.
  • the slots are preferably V-shaped. Accordingly, the standard will be stacked in the stacking gel and then, so to say, released into the resolving gel, while crossing the immobilized pH gradient strip (IGP); see Figures 2 and 4 for an illustration of the principle and the corresponding result.
  • IGP immobilized pH gradient strip
  • a grid is set up by the at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more samples of said standard.
  • the proteinaceous standard applied in the means and methods of the invention may be unstained or pre-stained.
  • the same molecular weight standard may have a different mobility and therefore a different apparent molecular weight when run in different buffer systems.
  • Each buffer system may have a slightly different pH which affects the charge of a protein and its binding capacity for, e.g., SDS. This can be pronounced in chemically modified proteins such as pre-stained standards and affect their apparent molecular weights significantly. Therefore, it is important to use the correct calibration values for the buffer system which is used.
  • Unstained standards may provide a better estimation of size than pre-stained standards.
  • pre-stained standards or pre-labeled standards are ideal and thus preferred for confirming the electrophoresis run.
  • Pre means that the standard is stained or labeled before its application in the means and methods of the invention.
  • the proteins of the standard may be modified and stabilized for use in electrophoresis. This often means that their apparent molecular weights may be different from that of the proteins in their native state. With pre-stained or pre-labeled standards, the difference between native and apparent molecular weights may be greater as proteins have been treated.
  • the proteinaceous standard is distinguishable from the label of the proteinaceous sample(s) that is subject to the methods of the invention. "Distinguishable” means that the proteinaceous standard does preferably not have the same label as one or more of the proteinaceous sample(s), thereby it is excluded that the proteinaceous standard interferes with the detection of a protein comprised by the proteinaceous sample or vice versa.
  • the proteinaceous standard is unstained, but nevertheless preferably distinguishable from the label of the proteinaceous sample(s).
  • the proteinaceous standard may be made visible by any known staining such as Coomassie or silver staining.
  • staining such as Coomassie or silver staining.
  • the separated, labeled proteins comprised by the proteinaceous sample are anyway detectable due to their label as described herein and can thus be recorded by a suitable means such as a digital photo.
  • the proteinaceous standard that is also separated can afterwards be detected by suitable means such as staining, for example, by Coomassie or silver staining.
  • a particular preferred proteinaceous standard meets the following criteria: molecular weights and/or pi of the proteins are distributed evenly across the gel proteins can be obtained in highly purified form
  • the mix can be stored in aliquots for longer periods of time
  • a more particularly preferred proteinaceous standard comprises the following proteins: Ubiquitin (bovine, 9 kDa, pi 6.8), Cytochrome C (horse, 12 kDa, pi 9.0), Trypsinogen (bovine, 24 kDa, pi 9.3), GAPDH (rabbit, 36 kDa, pi 8.4), and Albumin (bovine, 67 kDa, pi 5.5).
  • An even more particularly preferred proteinaceous standard comprises the following proteins: Ubiquitin (8.6 kDa), a-Lactalbumin (14.2 kDa), Myoglobin (17 kDa), Trypsin inhibitor (20.1 kDa), Trypsinogen (24 kDa), GAPDH (36 kDa), Ovalbumin (44 kDa), Albumin (69 kDa), and Phosphorylase b (97 kDa).
  • IEF isoelectric focusing
  • IPG immobilized pH gradient
  • a protein that is in a pH region below its isoelectric point (pi) will be positively charged and so will migrate towards the cathode. As it migrates through a gradient of increasing pH, however, the protein's overall charge will decrease until the protein reaches the pH region that corresponds to its pi. At this point it has no net charge and so migration ceases (as there is no electrical attraction towards either electrode). As a result, the proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pi. The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands.
  • Negatively charged molecules migrate through the pH gradient in the medium toward the "positive” end while positively charged molecules move toward the "negative” end.
  • a particle moves towards the pole opposite of its charge it moves through the changing pH gradient until it reaches a point in which the pH of that molecules isoelectric point is reached. At this point the molecule no longer has a net electric charge (due to the protonation or deprotonation of the associated functional groups) and as such will not proceed any further within the gel.
  • the gradient is established before adding the particles of interest by first subjecting a solution of small molecules such as polyampholytes with varying pi values to electrophoresis.
  • the proteinaceous sample is separated during isoelectric focusing preferably on a gel strip.
  • Said gel strip is then preferably polymerized in the stacking gel.
  • the stacking gel is equipped with wells for loading the standard; preferably these wells are V-shaped. V-shaping is preferably achieved by a V- shaped comb as described herein. A preferred comb is shown in Figures 3, 8, 9, 10.
  • the standard is subject to stacked gel electrophoresis, followed preferably transition-free by resolving gel electrophoresis.
  • the proteinaceous sample (separated on the gel strip) is simultaneously subject to resolving gel electrophoresis, i.e., together with the standard. Accordingly, during electrophoresis both the proteinaceous sample (separated on the gel strip) and the standard are separated. Thereby the proteins of the standard form a spot grid overlaying the separated proteins of the proteinaceous sample.
  • the proteinaceous sample is labeled. If more than one proteinaceous sample is subject to the methods as described herein, the proteinaceous samples are distinguishably labeled. For example, a first proteinaceous sample is labeled with a first label and a second proteinaceous sample is labeled with a second label, whereby the first and second label is different such that they are distinguishable. Similarly, the first and second label is different from a third label of a third proteinaceous sample, etc.
  • the proteinaceous sample may preferably be labeled prior to be subject of the methods of the invention. Less preferably, the proteinaceous sample may be labeled after the proteins contained in said proteinaceous sample are separated as described herein.
  • Labeling of a proteinaceous sample comprising, inter alia, proteins is done by means and methods commonly known in the art. Any suitable label can be used.
  • a preferred label is a cyanine dye.
  • Preferred cyanine dyes (such as Cy2, Cy3 and Cy5) are N-hydroxy succinimidyl ester derivatives. These cyanine dyes are typically covalently tagged to the ⁇ -amino group of lysine residue of proteins, and replace the ⁇ -amino group positive charge with the positive charge of the dye.
  • the binding of the dyes introduce small but matched increases in molecular weight to the protein. Since these dyes are hydrophobic, to prevent precipitation of proteins, preferably only one lysine residue per protein is labeled (minimal labeling).
  • the dyes are preferably all charge-matched and molecular mass-matched to prevent alterations of the isoelectric point and to minimize dye-induced shifting of labeled proteins during electrophoresis.
  • Another class of dyes that is also preferred are saturation dyes, which saturate cysteine residues instead of minimally labeling lysine residues. These dyes are also mass and charge-matched. Saturation cysteine dyes may have superior sensitivity to minimal lysine labeling.
  • one proteinaceous samples is first labeled with a first dye and the second proteinaceous sample with a second dye, for example, with Cy3 and then with Cy5.
  • the samples are subject to the methods of the invention.
  • the corresponding protein spot can be visualized by successively illuminating the gel with the excitation wavelengths of each of the dyes.
  • the resulting protein spots are analyzed using imaging software.
  • three dyes are applied, for example, to study multiple samples, proteinaceous samples are labeled with three dyes.
  • a third dye (such as Cy2) with similar characteristics as Cy3 and Cy5 may be used.
  • Proteinaceous samples are labeled, for example, with either Cy3 or Cy5, and a standard sample is labeled with Cy2. All samples are then applied to the same gel. After the run, the corresponding protein spot can be visualized by successively illuminating the gel with the excitation wavelengths of each of the dyes. The resulting protein spots are analyzed using imaging software. [0055] In the "comparing step" of the separation method of the invention the separated entities (preferably proteins of the proteinaceous sample) are compared with the internal standard, i.e., with the grid providing the coordinates of the proteins of the internal standard that are set in relation/compared to one or more (unknown) protein spots (of interest).
  • the internal standard i.e., with the grid providing the coordinates of the proteins of the internal standard that are set in relation/compared to one or more (unknown) protein spots (of interest).
  • a protein spot can be visualized by illuminating the gel with the excitation wavelengths of each of the dyes.
  • the resulting protein spots are analyzed using imaging software.
  • a protein spot is compared to the internal standard(s).
  • the internal standard provides a grid and thus landmarks which can be used as orientation points in the allocation of a protein spot to a distinct protein.
  • the relative position of a protein spot to a protein of the internal standard can be determined.
  • the grid provided by the internal standard can be reliably reproduced, it is possible to allocate each spot of a proteinaceous sample to a distinct protein. Also, by way of the means and methods of the present invention it is possible to run proteinaceous samples (clinical, environment or alimentary sample) collected days, months, or even years apart versus the same grid used for a reference profile from, e.g., clinics, environment or alimentary sources, since the grid generated by the internal standard remains (or will be) the same such that based on known relative values (i.e., relation between the position of the reference spot and position of a protein spot of interest) a protein spot of interest can be determined.
  • a protein spot that is relative to a protein of the internal standard, if of interest, can be picked and analyzed in order to determine the protein of the spot, for example, by its weight, fingerprint and/or sequence.
  • a database can be set up which compiles data of protein spots such as their relative position to a protein of the internal standard and, if more data about that protein spot are available such as the molecular weight or sequence of that protein, the database can also comprise that data.
  • a database can be generated for a typical proteinaceous sample, for example, from an environmental, clinical, or alimentary sample. By that, it is possible to compare an unknown proteinaceous sample to a proteinaceous sample characterized by the application of the means and methods of the invention.
  • the present invention relates to a method for identifying the presence and/or relative abundance of one or more proteins contained in a proteinaceous sample, which method comprises:
  • Relative abundance means that it is possible to determine the abundance of an analyte, preferably of a protein, for example in response to a certain stimulus, or depending on the stage of development of a cell; or simply depending on the sample as such, in a sample.
  • internal markers which allow a semi-quantitative determination of the abundance of an analyte (preferably of a protein).
  • Such internal markers are well-known and include, for example, the internal markers which are employed in the DIGE method/system as established by GE Healthcare.
  • the proteins spots of interest i.e. the proteins which are to be analyzed
  • ANOVA analysis of variance
  • the present invention relates to a method for determining the allergenic, carcinogenic, contamination or infection potential of a proteinaceous sample, which method comprises:
  • allergenic potential means the assessment of the potential of substances (preferably a proteins) to produce an allergic reaction in a human being or change their concentration in conjunction with allergy.
  • carcinogenic potential means the assessment of the potential of substances (preferably a proteins) to induce cancer in a human being or change their concentration in conjunction with cancer.
  • contamination potential means the assessment of the potential of a substance (preferably a protein) to contaminate a human being or the environment or change their concentration in conjunction with contamination.
  • infection potential means the assessment of the potential of a substance (preferably a protein) to infect a human being or change their concentration in conjunction with infection.
  • a substance contained in wheat flour that it has an allergenic potential.
  • this substance can be traced in other samples as a risk factor for a human being who is susceptible to an allergic reaction to suffer from an allergic reaction.
  • prion proteins have an infection potential. Accordingly, once identified in a proteinaceous sample that is subject to the means and methods of the present invention, the risk that this substance infects a human can be defined in other samples.
  • DIGE differential gel electrophoresis
  • 2D-DIGE is a method that labels protein samples with fluorescent dyes before 2D electrophoresis enabling accurate analysis of differences in protein abundance between samples.
  • DIGE internal gel electrophoresis
  • the internal standard is prepared by mixing together equal amounts of each sample of interest and including this mixture on every gel. The normalization of this internal DIGE-standard across gels allows the direct comparison of the ratio of relative abundance of the same protein in different samples. Nevertheless, as mentioned in the introduction part of this application, also the DIGE method suffers from several problems.
  • DIGE gels do not necessarily allow for a reliable and safe allocation of protein spots which are produced by one and the same protein on different gels, let alone a reliable and safe allocation of protein spots from a proteinaceous sample that is not run in parallel or simultaneously on the same gel. This is so, because the actual "running" behavior of a protein differs significantly when it is analyzed on different 2D-gels.
  • proteinaceous samples from different types of crops can be analyzed for their allergenic, carcinogenic, contamination or infection potential by the means and methods of the present invention.
  • proteinaceous samples are subject to the methods of the present invention and can be directly compared to each other because of their position in relation to the position of the reference spots of the grid (generated by the internal standard).
  • relative value(s) of the protein spots originating from said proteinaceous samples in relation (or in comparison) to the reference spots can be determined.
  • Such relative values can then be compared to known values from already run samples (preferably stored in a database). Such values may correspond to a protein spot known or suspected to be associated with an allergy.
  • a grid onto the DIGE-gel by way of (a) providing a proteinaceous standard of the invention; (b) subjecting said standard to stacked gel electrophoresis; and/or(c) subjecting said proteinaceous sample(s) and said standard to resolving gel electrophoresis.
  • step (c) is already the "standard" 2D-DIGE as such, with the only exception that (i) the proteinaceous standard of the present invention is employed in addition to the DIGE standard and (ii) that the 2D-DIGE gel comprises a further stacking gel which allows the focusing and concentration of the proteinaceous standard of the present invention in order to result in a grid on the actual 2D-DIGE gel (the stacking gel is the one which was already mentioned in the above defined step (b)).
  • the internal DIGE standard is differently labeled in regard to the remaining samples that are to be analyzed (preferably all these samples are labeled by a different fluorescent label).
  • the internal DIGE standard may be seen as a mere further proteinaceous sample which enables the normalization of differentially expressed to a standard value.
  • the DIGE standard does not allow any prediction of the different proteins as such (i.e., it is not possible to use the DIGE standard for the establishment of a grid which allows the precise localization of different protein spots on different gels, thereby facilitating a reliable and fast analytic system which is even automatable, if desired).
  • the present invention relates to a 2D difference fluorescence gel electrophoresis (DIGE) method, characterized in that a DIGE separated protein is additionally compared to a standard as defined in any one of the preceding claims in order to identify said DIGE separated protein.
  • DIGE 2D difference fluorescence gel electrophoresis
  • the mentioned standard denotes a proteinaceous standard of the present invention. It will likewise be understood that this standard is preferably applied by way of a stacking gel and in accordance with the principles explained herein above.
  • the present invention also relates to a DIGE/2D-DIGE system/method (provided by GE Healthcare), which method is modified and/or combined with the methods of the present invention.
  • the DIGE method is accompanied by the step of (a) providing a proteinaceous standard of the invention; (b) subjecting said standard to stacked gel electrophoresis; and/or (c) subjecting said proteinaceous sample(s) and said standard to resolving gel electrophoresis, wherein said resolving gel electrophoresis is a 2D-DIGE gel (optionally including the internal DIGE standard).
  • the present invention relates to the gel of the invention as such, namely to a gel (suitable) for gel electrophoresis of proteins comprising (from top to down):
  • “Top” means that side of the gel which comprises the open side of the slots of the stacking gel (i.e. the stacking gel which focuses and concentrates the proteinaceous standard of the present invention). It is shown in Figure 2 that an additional stacking gel is polymerized on top of the regular 2D-gel containing the pl-strip and a comb as used in 1 D-PAGE. This assembly was termed gel-strip-sandwich (GSS).
  • GSS gel-strip-sandwich
  • the above mentioned stacking gel comprises one or more V-shaped slots.
  • One preferred embodiment of such a gel is schematically depicted in Figure 2, although the present invention is in no way limited to this scheme. The skilled person is well aware that there are many possible ways to alter this scheme - the only important thing is that all the above gel parts are arranged in the above indicated order (stacking gel, containing pH gradient gel, then resolving gel).
  • the predetermined pH gradient gel may already comprise one or more proteinaceous samples of the present invention.
  • the stacking gel already comprises a proteinaceous standard of the present invention.
  • It is also envisaged to provide the gels in a pre-assembled fashion for example in sealable bags comprising the stacking gel, the pH gradient gel and/or the resolving gel).
  • Other examples include sealable or sealed bags or containers which comprise the stacking gel and the resolving gel (preferably separated) and an imprint or technical instructions which will instruct the skilled person to provide a pH gradient gel and to employ the pH gradient gel in accordance with the gist of the present invention.
  • Figure 4 shows a resulting gel with a standard mixture (1D Precision Plus Dual Color Standards, Bio-Rad). Across the gel are five lanes corresponding to the five V-wells which contained the protein standard. It is obvious that the proteins became more focussed and that a spot-like appearance could be achieved. Also, a grid has been generated.
  • the present invention relates to the gel and/or the methods and/or the uses of the present invention (as defined herein), wherein said stacking gel comprises at least one V-shaped slot.
  • V-shaped means that at least the lower part of the slot (i.e. the part which points towards the resolving gel) is shaped in the form of the letter "V". This means, however, that the remainder of the slot may have a different form (for example the form of a square - see Figure 3).
  • the angle of the "V is preferably below 90°, 80°, 70°, more preferably below 60°, or 50° and even more preferred below 45°, 40° or 30°. Most preferred is an angle of about 28°. It is to be understood that the terms “below” or “about” include the value as such.
  • V-shaped combs represent one non-limiting way to incorporate V-shaped slots in the stacking gel (it is, for example, also envisaged to cut the correct form into the polymerized gel). These combs can be provided without further ado. It will be appreciated that it is possible to optimize the angle of the "V” in regard to the specific stacking gel that is to be used. This means that dependent on the composition of the stacking gel (it is for example standard to vary the mesh size of the stacking gel by way of employing different concentration of for example polyacrylamide) a different angle might lead to a more "focused" appearance of the resulting spots (see Figure 5).
  • the present invention also provides a comb that is preferably for use in the methods of the present invention. However, it can also be contained in the kits and gels described herein.
  • the comb has preferably at least one shark-tooth, more preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more shark-teeth.
  • a shark-tooth has a triangular shape.
  • the height of a shark-tooth is preferably about 10 mm.
  • the angle at the apex of the triangle has preferably about 28°, while the remaining two angles have preferably about 76° (each).
  • the distance between two shark-teeth is preferably about 4 mm.
  • the distance between an apex of a first triangle and the apex of a second triangle is preferably about 9 mm (see Figure 8), however, any other distance is also possible, i.e. smaller than 9 mm or larger than 9 mm, such as 5, 6, 7, 8 or 10, 11 , 12, 13, 14, 15 mm.
  • the thickness of the comb is variable.
  • a comb may have a thickness of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4 or 1.5 mm.
  • the thickness of the comb preferably directly corresponds to the thickness of the gel.
  • the thickness of the comb is preferably about 1 mm (see Figure 10).
  • a preferred comb with shark-teeth has the measures as shown in Figure 8.
  • the comb combines triangular and rectangular teeth, thereby providing a choice of generating V-shaped and pocket-shaped wells or only V-shaped wells.
  • a preferred comb with combined triangular and rectangular teeth has the measures as shown in Figure 9.
  • the comb may be molded from any suitable plastic including, but not limited to polymers such as polyethylene terephthalate, polyvinyl chloride, polymethyl methacrylate, polystyrene, polyethylene, polyethylene, polypropylene, cellulose acetates, co-polymers, polycarbonate, or any other suitable material, with polycarbonate being preferred.
  • said stacking gel comprises polyacrylamide in a concentration which is sufficient to concentrate the proteinaceous sample of the present invention in a spot-like manner, thereby leading to a grid-structure on the gel of the present invention (as explained herein elsewhere).
  • the present invention also relates to a kit comprising at least one or even all of:
  • Said kit may further comprise package insert and/or instructions comprising instructions on how to use the gels in accordance with the methods of the present invention.
  • the term "package insert and/or instructions' is further used to refer to instructions customarily included in commercial packages of research products, that contain information about the methods, usage, storage, handling, and/or warnings concerning the use of such products.
  • the kits of the present invention may further comprise positive and/or negative controls and/or a data carrier which encompasses a computer program of the present invention.
  • the present invention relates to the use of a gel or kit defined herein, for determining the allergenic, carcinogenic, contamination or infection potential of a proteinaceous sample and/or for identifying the presence of one or more proteins contained in a proteinaceous sample and/or for separating a plurality of proteins.
  • a gel or kit defined herein for determining the allergenic, carcinogenic, contamination or infection potential of a proteinaceous sample and/or for identifying the presence of one or more proteins contained in a proteinaceous sample and/or for separating a plurality of proteins.
  • a master gel depicts a pre-determined grid, i.e., a pre-determined proteinaceous standard is used.
  • Pre-determined means that the proteinaceous standard is assembled such that it provides a grid which reflects the needs of the actual analysis - for example: if protein samples are to be examined which contain proteins having a molecular weight of about 10 to 50 kDa, then the proteinaceous standard of the invention should preferably also contain protein markers having a comparable size (10 to 50 kDa).
  • the master gel might help to enhance the analysis and recovery of grid spots on the gels of the invention (it will be understood that these gels should make use of the very same proteinaceous standard which was already employed for the master gel).
  • "Compare” simply means that the grid data obtained with the master gel and the grid data obtained with the gels as such (i.e. the gels with which the samples are analyzed) are compared with each other in order to aid the skilled person and/or the software/computer which is used for the analysis of the gels to relocate the grid spots in the gels with which the samples are analyzed.
  • the invention provides a system for determining the allergenic, carcinogenic, contamination or infection potential of a proteinaceous sample and/or for identifying the presence of one or more proteins contained in a proteinaceous sample and/or for separating a plurality of proteins, said system comprising: a) an analyzer for analyzing the grid and/or the protein spots on the gels of the invention; and optionally b) a comparator for comparing the grid and/or the protein spots measured by the analyzer with a master gel.
  • the above system is preferably a computer system comprising a processor and a data storage device or computer readable medium, having stored thereon data of one or more master gels (in particular the grid data of these master gels), and optionally the data of one or more gels which gels display one or more samples described herein above, wherein optionally the system further comprises a comparison algorithm, or, optionally further comprises an identifier that identifies one or more features in the gels, and optionally the comparison algorithm comprises a computer program that indicates differences between different samples on different gels or different samples on the same gel or between samples displayed by a gel of the invention in comparison to a master gel of the invention.
  • An "identifier" refers to one or more programs which identifies certain features within a gel or between gels. Said features include but are not limited to the grid, the spots which are analyzed in the respective samples (preferably the proteinaceous samples), the label with which the standards and samples are labeled, and/or the gel as such.
  • a computer system refers to the hardware components, software components and data storage components used to analyze the gels of the invention (which includes the master gels of the invention).
  • a skilled artisan can readily appreciate that any one of the currently available computer systems are suitable.
  • the invention provides a memory device for storing data for access by an application program being executed on a data processing system for identifying protein spots on a gel of the invention (either as such or by way of comparing the data with a master gel or data obtained with a master gel), said memory comprising: a data structure stored in said memory, the data structure including information resident in a database used by said application program and including grid and/or protein data confirming the presence and/or location of a protein (and/or grid) on a 2D gel.
  • the present invention further relates to a computer program capable of conducting the identification/determination as defined herein above. Said computer program may be used in the above mentioned system of the invention.
  • Figure 1 General procedures in 2D-PAGE taking advantage of protein properties.
  • the sample mixture is first separated on a small strip. Then the strip is placed on top of a blank polyacrylamide gel and the proteins move into the gel. For orientation a marker mixture can be applied in addition.
  • Figure 2 Side view of a gel assembly for the production of Grid-Gels. An additional stacking gel holds the pl-strip as well as a comb to generate sample wells (GSS-PAGE).
  • FIG. 3 Combs as they are typically used for 1D-PAGE were modified to provide
  • FIG. 4 Generation of a protein grid on a 2D-gel by use of V-wells and a rehydrated blank pl-strip in a GSS assembly. As a marker, 1D Precision Plus Dual Color Standards were used and Coomassie stained.
  • FIG. 5 Generation of sharp focused spots of Grid-Mix by using V-wells in 1D- PAGE.
  • Grid-Mix Protein Standard Ubiquitin (bovine, 9 kDa, pi 6.8);
  • Cytochrome C (horse, 12 kDa, pi 9.0); Trypsinogen (bovine, 24 kDa, pi 9.3); GAPDH (rabbit, 36 kDa, pi 8.4); Albumin (bovine, 67 kDa, pi 5.5).
  • Figure 6 1 D-PAGE of individual standard proteins for Grid-Mix.
  • Figure 7 Generation of a protein grid in repeated experiments by use of V-wells.
  • FIG. 8 Comb with shark-teeth. Upper panel shows the left half of a preferred comb of the present invention, while the middle panel shows the right half. The lower panel shows a half of a preferred comb having on one side shark-teeth and on the other side combined triangular and rectangular teeth. All measures are in millimeter.
  • Figure 9 Comb with combined triangular and rectangular teeth. Upper panel shows the left half of a preferred comb of the present invention, while the middle panel shows the right half. The lower panel shows a half of a preferred comb having on one side shark-teeth and on the other side combined triangular and rectangular teeth. All measures are in millimeter.
  • Figure 10 Segment of a preferred comb showing a preferred width of a comb described herein.
  • Figure 11 Cy3 image of Grid-Mix (left) and Cy5 image of wheat flour obtained in a
  • Comparative fluorescence gel electrophoresis was introduced to improve comparability of 2D gel electrophoresis (2D) experiments.
  • the technology is a combination of 1 D- and 2D-PAGE (gel-strip-sandwich GSS) and is based on fluorescent labelling of proteins to allow several samples to be separated on a single gel.
  • a V-well- forming gel comb is placed above the pl-strip containing separated proteins.
  • the wells are used for the application of solutions of marker proteins.
  • both the proteins from the pl-strip and the proteins in the wells are separated according to their molecular weight. Thereby, the marker proteins form a spot grid overlaying the separated proteome sample and providing anchor spots for reproducible assignment of their spot coordinates.
  • a preferred step-by-step protocol for this methodology is presented below.
  • Protein concentrations were determined using a spectrophotometer (Ultrosec 2000, Pharmacia Biotech) at 590 nm. The pH value of the sample was adjusted to 8-9 at 4 °C with dilute HCL or NaOH solution and was determined with a pH meter (Orion PerpHect ® pH Electroden 8220 from ThermoFisher). Samples were labelled with Cy3, Cy5 or Cy2 according to manufacturer ' s instructions (GE Healthcare). For isoelectric focusing, 24 cm DryStrips pH 3-10 were used. The second dimension was run at 20 °C at 15 % resolving gel and 4 % stacking gel.
  • the gels were scanned directly or after being stored at 4 °C overnight with Typhoon 9400 variable imager using the blue laser (488 nm) for Cy2 (emission filter 520 BP 40), the green laser (532 nm) for Cy3 (580 BP 30) and the red laser (633 nm) for Cy5 (670 BP 30).
  • Protein standards (ubiquitin from bovine red blood cell, a-lactalbumin from bovine milk, myoglobin from horse heart, trypsin inhibitor from glycine max soybean, trypsinogen from bovine pancreas, GAPDH from rabbit muscle, ovalbumin from chicken egg, albumin from bovine, phosphorylase b from rabbit muscle) were purchased from Sigma-Aldrich. Stock solutions were prepared, if deemed necessary. Suitable mixtures of all proteins were dried and stored at -20 °C. Such aliquots are stable for at least six months. Prior to use they are dissolved in 200 ⁇ DIGE lysis buffer (30 mM Tris, 8 M urea, 4% (w/v) CHAPS, pH 8.5).
  • GSS-PAGE experiments using Cy dyes include preferably maximal two samples and one protein standard mix for grid formation.
  • the marker proteins in the standard mix should have distinct molecular weights covering the measurement range and should focus well as a spot in 1-DE using V-wells.
  • Protein standard ubiquitin, a-lactalbumin, myoglobin, trypsin inhibitor, trypsinogen, GAPDH, ovalbumin, albumin, phosphorylase b
  • Stock solutions are dissolved prior to use in 200 ⁇ lysis buffer 2 (Recipe 1 ).
  • the buffer used for protein lysation or solubilisation should not contain DTT, respective thiols, primary amines or ampholytes.
  • the salt concentration should not exceed 30 mM at pH 8.5 (4 °C).
  • the pH of the protein solutions also needs to be readjusted to pH 8-9.
  • the sample volume prepared for loading onto the pl-strip should not exceed 65 ⁇ , because the IEF loading cup can hold only 150 ⁇ , the sample has to be diluted 1 :2 with 2X sample buffer (Recipe 2, 3) and 20 ⁇ cover oil needs to be added to the cup to avoid urea crystallization and contact with oxygen.
  • DMF must be of high quality ( >99.8 %; ⁇ 0.005 % H 2 0). It is recommended to open a new DMF bottle every 3 months. Bring the closed dye vials to room temperature for 5 min. Add 5 ⁇ DMF to 5 nmol dye. Vortex vigorously for 30 s to dissolve the dye. Centrifuge for 30 s at 13.000 RPM. The dye stock solution (1 nmol/ ⁇ ) is stable for 3 months at -20 °C in a light-excluding tube.
  • Recipe 5 Quenching solution. It is stable for 6 month at -20°C.
  • Resolving gels have a size of 25.5 cm x 19 cm x 1 mm.
  • the required height of the stacking gel is ⁇ 1.5-2 cm. It is important to use low fluorescence glass plates without scratches in order to achieve high quality images with low background.
  • the gel caster is prepared as recommended by the manufacturer with the important difference that ⁇ 2 cm on top of each gel cassette should remain free of gel solution. It is somewhat tricky to achieve identical heights for the six resolving gels in the caster. In that respect the use of commercial gels may be of advantage.
  • Tris-HCI 54.5 g Tris, 200 ml MilliQ-water). Adjust pH to 8.8 with 6 M HCI. Make up the volume to 300 ml with MilliQ-water. The solution is stable for 1 month at 4 °C.
  • Electrophoresis buffer (anode buffer)
  • equilibration buffer I and II Prior to use. Take out the strip out onto a filter paper with the gel side up to remove residual cover fluid. Were strips stored at -20 °C, bring them to room temperature for 10 min. Place the strips in individual equilibration tubes with the gel side up. Add 10 ml equilibration buffer I to each equilibration tube. Incubate the strips for 15 min at room temperature with gentle agitation. Longer equilibration times are not advisable to avoid protein diffusion. During equilibration prepare the stacking gel solution. Mix the components except TEMED and 10 % APS. After stirring for 15 min with magnetic stirrer, replace equilibration buffer I with equilibration buffer II and incubate for 15 min at room temperature with gentle agitation. Recipe 10-12)
  • Recipe 12 4 % Stacking gel solution. Prepare fresh solution prior to use.
  • Reagent 10 ml 20 ml 30 ml 40 ml 50 ml 60 ml
  • the protein standard was premixed and dried using SpeedVac at 35 °C. It is stable for at least 6 months at -20 °C. Just prior to use, dissolve an aliquot in 200 ⁇ lysis buffer 2. To generate sufficient marker solution for 100 lanes, 67 ⁇ (65 pg total protein) is labelled with 520 pmol Cy3 (1.3 pi of 400 pmol/ ⁇ dye solution). Labelling is quenched with 1.3 pi 10 mM lysine [2]. The labelled mix is diluted with 130.4 ⁇ Laemmli buffer 2 and 2 ⁇ are applied to each lane (Recipe 13).
  • the recommended ratio of dye to protein is 8 pmol dye / 1 pg protein.
  • the loading amount of each protein standard in each well can be maximal 2 ⁇ .
  • the labelled mix should be diluted at minimum 1 :2 with Laemmli buffer 2. At this protein/dye ratio, the volume of Cy3 working solution can vary a little. Consequently the volume of Laemmli buffer varies to some extent. The total volume of the mix after dilution must be 40 ⁇ .
  • the second dimension was run at 20 °C at 15 % resolving gel and 4 % stacking gel.
  • Example 2 1. Materials and methods
  • Protein concentrations were determined using a spectrophotometer (Ultrospec 2000, Pharmacia Biotech) at 590 nm. Sample pH was adjusted to 8-9 at 4 °C with dilute HCI or NaOH solution and was determined with a pH meter (Orion PerpHect ® pH electrode 8220, ThermoFisher). Samples were labelled with Cy3, Cy5 or Cy2 according to manufacturer's instructions [2]. The second dimension using 15 % resolving gel and 4 % stacking gel was run at 20 °C.
  • a Comparative Fluorescence Gel Electrophoresis (CoFGE) set-up was designed which unified elements of 1- and 2-DE.
  • 1-DE protein solutions are applied to sample pockets in the polyacrylamide gel and separated during electrophoresis according to their molecular weight.
  • 2-DE proteins are pre-separated on a pi strip based on their isoelectric point. This strip is then placed on top of the resolving gel for electrophoresis.
  • CoFGE combines the use of pockets and pi strips in a single run facilitating a 1-DE experiment in parallel to 2-DE separation. Thereby, the latter separates the sample proteome while the former generates anchor points for reproducible determination of protein coordinates.
  • V- teeth can alternate with rectangular teeth depending on the separation task.
  • Wells can be formed up to 10 mm in height allowing the loading of maximal 25 ⁇ of sample into V- wells and 70 ⁇ into pocket wells.
  • the use of low sample volumes improved protein spot size and shape.
  • a volume of 2 ⁇ is recommended.
  • V-wells have therefore the additional advantage when used in regular 1-DE that the proteins are better focussed, leading to an increase in sensitivity.
  • the V-well comb was designed with a flexible holder with graduation which could be adjusted in height.
  • the entire assembly was called Gel-Strip-Sandwich (GSS).
  • the size of the resolving gel was 25.5 cm (w) x 19 cm (h) and the stacking gel was 2 cm high. Skill had to be acquired to generate GSS without trapping air bubbles.
  • a 4 % stacking gel was optimal for this application; at higher gel density, the protein standard mix applied into the wells did not exhibit the same running front as a protein with the same molecular weight in the pi strip. Polymerization was performed for 45-60 min before application of the marker proteins to the V-wells. One hour setting time should not be exceeded, because the samples loaded on the pi strip may diffuse with a negative effect on resolution.
  • the mix was dissolved in 200 ⁇ DIGE lysis buffer 2 (30 mM Tris, 8 M urea, 4% (w/v) CHAPS, pH 8.5). To generate sufficient marker solution for 100 lanes, 67 ⁇ (65 g total protein) was labelled with 520 pmol Cy3 (1.3 ⁇ of 400 pmol/ ⁇ dye solution). The labelling was quenched with 1.3 ⁇ 1 10 mM lysine [2]. The labelled mix was diluted with 130.4 ⁇ Laemmli buffer 2 and 2 ⁇ was applied to each lane.
  • V-well combs were tested without pl-strip (experiments with a modified commercial comb) and later with a blank pl-strip (Figure 4).
  • a commercial protein standard was first used for this purpose (1 D Precision Plus Dual Color Standards, Bio- Rad). Across the gel five lanes corresponding to the five V-wells were formed. Proteins were better focussed and a spot-like appearance was achieved. Spot shape was dependent not only on teeth shape but also on sample volume and running parameters. It proved very effective to dilute the protein mixture with Laemmli buffer containing 50 % glycerol instead of 25 % (v/v) glycerol. This measure increased the density of the protein mix solution and secured the samples at the bottom of the wells.
  • FIG. 11 An example is shown in Figure 11 for the separation of a protein extract of whole wheat flour, since this proteome was of particular interest for studies with respect to baker's allergy.
  • the Grid-Gel does not correct for subtle changes in the pi of the sample.
  • pl-adjusted protein mixtures with a third dye to control the first dimension. We have tested this and did not find this measure necessary for our purposes, in particular, when manual control of spot assignment was performed.
  • the IEF process worked very reliably and the sample proteins reproducibly found their position on the pl-strip.
  • a 2-DE set-up is presented which allows the generation of a protein spot grid in the same gel that carries the proteome to be investigated. This is possible by use of charge- and mass-matched fluorescent dyes such as Cy dyes. Cy3 and Cy5 were typically chosen for the grid and the sample, respectively, since they have similar sensitivity.
  • a special gel comb with V-shaped teeth was designed and placed in a stacking gel above the pi strip. This design allowed the generation of, e.g., grids with 9 lanes and 13 columns, where the nodes served as anchor points for the determination of protein spot coordinates of the analyte sample located on the same gel. This technology promises to solve some of the issues related to gel comparison over time and among laboratories.
  • V-well combs are also of advantage to create better focussed protein spots in regular 1-DE, e.g. for improved sensitivity or marking purposes.
  • Well distances and comb parameters can be freely chosen within the limits of the respective gel chamber. The methodology does not require extensive instrumentation or special chemicals. For moderate cost GSS-PAGE or CoFGE can be set-up in any laboratory.
  • Table 4 Composition of reference protein mixture (Grid-Mix) aqueous stock solutions. Protein standards were purchased from Sigma-Aldrich. The mixture was dried using a SpeedVac (Savant SpeedVac ® , ThermoFisher; 35°C) and stored at -20 °C. It was stable for at least 6 months.
  • proteomes based on 2-DE are often required in biochemical and clinical research.
  • DIGE technique is widely used [1-4]. Thereby, up to three samples are labeled with a suitable fluorescent dye. All three samples are run on a single gel and a fluorescence scanner separates the individual images. This method has eliminated many problems associated with gel-to-gel variation and also provides statistically evaluated protein ratios.
  • V-well combs were introduced.
  • Commercially available combs as they are typically used to generate wells for sample application in 1-DE, were modified.
  • Their rectangular teeth were cut to form a V-shape [8] with the goal of reducing the width of the resulting protein band and approaching spot size.
  • the shape of the teeth was shown to be important; the sharper the teeth, the smaller the protein spot.
  • tooth shape had an influence on well stability. Pointed tips have been used in DNA gel electrophoresis (shark teeth [9]), but these combs had different dimensions being used for ultra-thin gels and were not suitable for our purpose.
  • V-wells had the additional advantage when used in regular 1-DE that proteins were better focussed leading to an increase in sensitivity. This was however only true for gels of regular size (-25x19 cm), not for minigels (-8x7 cm), because gel size and run time were shown to be important for the formation of spots [10]. Conclusively, CoFGE does not function at its full capacity on minigels.
  • the CoFGE gel assembly is shown in Fig. 2.
  • an additional 4% stacking gel was polymerized on top of the regular 2-DE 15% resolving gel immediately after pH-strip insertion.
  • the stacking gel solution contained Bromopenol blue for both the visualization of the running front and the sample wells for loading; they would be almost invisible otherwise.
  • a 28-teeth V-well comb was inserted into the stacking gel analogous to 1-DE. Care had to be taken not to damage the pl-strip in the process and a distance of -2 mm from tip of teeth to upper side of strip was recommended. The comb must not touch the strip, otherwise spot shape suffered.
  • the V-well comb was designed with a flexible holder with graduation, which could be adjusted in height.
  • the entire assembly was called Gel-Strip-Sandwich (GSS).
  • the size of the resolving gel was 25.5 cm (w) x 19 cm (h) and the stacking gel was approximately 2 cm high.
  • a 4% stacking gel was optimal for this application; at higher gel density, the protein standard mix applied into the wells did not exhibit the same running front as a protein with the same molecular weight in the pi strip. Even for a 4% gel matrix, marker spots did not exactly co-localize in test runs, but this percentage was the limit - otherwise V-wells did not polymerize properly. Polymerization was performed for only 15 min before application of the marker proteins to the V-wells.
  • V-well combs were tested without or with a blank pl-strip using commercial protein standard mixtures (1D Precision Plus Dual Color Standards, Bio-Rad) and visible stains.
  • the buffer composition of such mixtures was not compatible with fluorescent staining so that we combined individual proteins to form a reference protein mixture suitable to generate about equally stained spots for the molecular weight range from 8.6 to 97 kDa on a 15% polyacrylamide gel (Grid-Mix, Suppl. Table S1 ).
  • Fig. S1A shows the grid images for all six gels including the edges of the glass plates to provide a general impression of the data output. Thirteen V-wells were loaded except in gel 4, where the first V-well was damaged. Looking at all grid images it was immediately obvious that no two were identical despite parallel processing. Polymerization effects played a larger role as anticipated in this setting and the grid image visualized this effect much more drastically then a sample spot image did.
  • Fig. S1B shows exemplary cropped images of grid and the corresponding proteome sample for gel 5. It was images like these which were uploaded to Delta 2D software for analysis. For completeness sake, Fig. S1C presents false colour overlays of grid and proteome for all six gels.
  • the Grid-Gel does not correct for subtle changes in the pi of the sample.
  • pl-adjusted protein mixtures with a third dye to control the first dimension. We have tested this and did not find this measure necessary for our purposes.
  • the IEF process worked very reliably and the sample proteins reproducibly found their position on the pl-strip, but the potential influence of pi needs to be kept in mind.
  • Protein concentrations were determined using a spectrophotometer (Ultrospec 2000, Pharmacia Biotech) at 590 nm. Sample pH was adjusted to 8-9 at 4°C with dilute HCI or NaOH solution and was determined with a pH meter (Orion PerpHect® pH electrode 8220, ThermoFisher). Samples were labelled with Cy3, Cy5 or Cy2 according to the manufacturer's instructions [1] (with 2M lysine for quenching [2]). The second dimension using 15% resolving gel and 4% stacking gel was run at 20°C.
  • E. coli lyophilisate (25.5mg, Sigma EC11303-10G) was dissolved in 1 ml lysis buffer (30 mM Tris, 8 M urea, 4% (w/v) CHAPS, pH 8.5). After 30 min shaking on a horizontal shaker (1500 rpm, room temperature) the sample was centrifuged for 10 min (12.000 x g; 4°C). The supernatant was transferred in a new tube; the pellet was discarded.
  • Table S1 Composition of reference protein mixture (Grid-Mix) aqueous stock solutions. The mixture was dried using a SpeedVac (Savant SpeedVac®, ThermoFisher; 35°C) and stored at -20°C. It was stable for at least 6 months. This mix can be individually optimized in concentration and composition to generate spots according to need, possibly for higher or lower molecular weights. Cytochrome C, in principle a well suited pure calibration protein (still used in Fig. 3), was excluded because it exhibited self- fluorescence.
  • the mix was dissolved in 200 ⁇ DIGE lysis buffer 2 (30 mM Tris, 8 M urea, 4% (w/v) CHAPS, pH 8.5).
  • DIGE lysis buffer 2 30 mM Tris, 8 M urea, 4% (w/v) CHAPS, pH 8.5.
  • 67 ⁇ 65 ⁇ g total protein
  • Cy3 1.3 ⁇ of 400 pmol/ ⁇ dye solution
  • the labelling was quenched with 1.3 ⁇ 10 mM lysine [1].
  • the labelled mix was diluted with 130.4 ⁇ Laemmli buffer 2 and 2 ⁇ was applied to each lane.
  • Phosphorylase b rabbit muscle a 97 10.3 I 22.5 22.5 0.075
  • Table S2 Mean and deviation from mean in percent for coordinates of selected protein fate in CoFGE gels of £ cali lysate before and after waiping against the marker grid. Data C1-C far nnwarped gels is available in the Snpplementary Excel file.

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Abstract

L'invention concerne un procédé de séparation de plusieurs protéines, lequel procédé consiste à fournir un échantillon protéiné étiqueté ou plusieurs échantillons protéinés étiquetés de manière distinguable ; fournir une norme protéinée qui est distinguable de ladite étiquette du ou des échantillons protéinés ; soumettre le ou les échantillons protéinés à une focalisation isoélectrique dans un gel à gradient de pH ; soumettre la norme à une électrophorèse par gel en empilement, soumettre le ou les échantillons protéinés et ladite norme à une électrophorèse par gel de résolution ; et comparer les entités protéinées séparées et la norme interne séparée. Ces procédés peuvent également être utilisés pour identifier la présence d'une ou de plusieurs protéines contenues dans un échantillon protéiné, ou afin de déterminer le potentiel allergène, carcinogène, de contamination ou d'infection d'un échantillon protéiné. L'invention concerne également un gel convenant pour l'électrophorèse par gel de protéines qui comprend (du haut vers le bas) un gel d'empilement, un gel à gradient de pH prédéterminé, et un gel de résolution.
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CN109239170A (zh) * 2018-08-24 2019-01-18 浙江理工大学 一种基于蛋白质组学鉴别古代毛织品残片毛种类的方法
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Publication number Priority date Publication date Assignee Title
JP2016525700A (ja) * 2013-07-01 2016-08-25 ライカ ミクロジュステムス ツェーエムエス ゲーエムベーハー レーザ顕微切離システム及び核酸含有試料の検査方法
CN109239170A (zh) * 2018-08-24 2019-01-18 浙江理工大学 一种基于蛋白质组学鉴别古代毛织品残片毛种类的方法
CN109239170B (zh) * 2018-08-24 2020-09-29 浙江理工大学 一种基于蛋白质组学鉴别古代毛织品残片毛种类的方法
CN116218009A (zh) * 2023-03-08 2023-06-06 浙江纽瑟生物技术有限公司 一种用于蛋白质电泳在线显色胶的制胶胶板及其制备方法

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