In-gel digestion
Encyclopedia
The in-gel digestion is part of the sample preparation
Sample preparation (mass spectrometry)
The sample preparation for mass spectrometry is used for the optimisation of a sample for the analysis in a mass spectrometer .These have depending on their ion source different requirements for volume, concentration, and composition of the analyte solution...

 for the mass spectrometric
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...

 identification of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s in course of proteomic analysis
Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...

. The method was introduced 1992 by Rosenfeld. Despite innumerable modifications and improvements the basic elements of the procedure remain largely unchanged.

The in-gel digestion primarily comprises the four steps destaining, reduction
Redox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....

 and alkylation
Alkylation
Alkylation is the transfer of an alkyl group from one molecule to another. The alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion or a carbene . Alkylating agents are widely used in chemistry because the alkyl group is probably the most common group encountered in...

 (R&A) of the cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...

s in the protein, proteolytic cleavage of the protein and extraction
Extraction (chemistry)
Extraction in chemistry is a separation process consisting in the separation of a substance from a matrix. It may refer to Liquid-liquid extraction, and Solid phase extraction....

 of the generated peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...

s.

Destaining

Proteins which were separated by 1D or 2D PAGE
Page
-Position or occupation:* Page , a traditionally young male servant* Page * Page of Honour, a ceremonial position in the Royal Household of the Sovereign of the United Kingdom* A participant in any of the following programs:...

 are usually visualised by staining with dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....

s like Coomassie Brilliant Blue (CBB) or silver. Although the sensitivity of the method is significantly lower, the use of Coomassie is more common for samples destined for mass spectrometry since the silver staining impairs the analysis. After excision of the protein band of interest from the gel most protocols require a destaining of the protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s before proceeding.

The destaining solution for CBB contains usually the buffer
Buffer solution
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...

 salt
Salt
In chemistry, salts are ionic compounds that result from the neutralization reaction of an acid and a base. They are composed of cations and anions so that the product is electrically neutral...

 ammonium bicarbonate
Ammonium bicarbonate
Ammonium bicarbonate, a compound with formulaNH4, also called bicarbonate of ammonia, ammonium hydrogen carbonate, hartshorn, AmBic or powdered baking ammonia, is the bicarbonate salt of ammonia....

 (NH4HCO3) and a fraction of 30%-50% organic
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...

 solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...

 (mostly acetonitrile
Acetonitrile
Acetonitrile is the chemical compound with formula . This colourless liquid is the simplest organic nitrile. It is produced mainly as a byproduct of acrylonitrile manufacture...

). The hydrophobic interactions between protein and CBB are reduced by the organic fraction of the solution. At the same time diminishes the ionic part of the solution the electrostatic
Electrostatics
Electrostatics is the branch of physics that deals with the phenomena and properties of stationary or slow-moving electric charges....

 bonds between the dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....

 and the positively charged
Electric charge
Electric charge is a physical property of matter that causes it to experience a force when near other electrically charged matter. Electric charge comes in two types, called positive and negative. Two positively charged substances, or objects, experience a mutual repulsive force, as do two...

 amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

s of the protein. In contrast to a mixture of water
Water
Water is a chemical substance with the chemical formula H2O. A water molecule contains one oxygen and two hydrogen atoms connected by covalent bonds. Water is a liquid at ambient conditions, but it often co-exists on Earth with its solid state, ice, and gaseous state . Water also exists in a...

 with organic solvent the effectivity of destaining is increased. An increase of temperature
Temperature
Temperature is a physical property of matter that quantitatively expresses the common notions of hot and cold. Objects of low temperature are cold, while various degrees of higher temperatures are referred to as warm or hot...

 promotes the destaining process. To a certain degree (< 10%) the destaining procedure is accompanied with a loss of protein. Furthermore, the removal of CBB does not affect the yield of peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...

s in the mass spectrometric measurement.

In the case of silver stained protein bands the destaining is accomplished by oxidation of the metallic silver
Silver
Silver is a metallic chemical element with the chemical symbol Ag and atomic number 47. A soft, white, lustrous transition metal, it has the highest electrical conductivity of any element and the highest thermal conductivity of any metal...

 attached to the protein by potassium ferricyanide
Potassium ferricyanide
Potassium ferricyanide is the chemical compound with the formula K3[Fe6]. This bright red salt contains the octahedrally coordinated [Fe6]3− ion. It is soluble in water and its solution shows some green-yellow fluorescence.-Preparation:...

 or hydrogen peroxide
Hydrogen peroxide
Hydrogen peroxide is the simplest peroxide and an oxidizer. Hydrogen peroxide is a clear liquid, slightly more viscous than water. In dilute solution, it appears colorless. With its oxidizing properties, hydrogen peroxide is often used as a bleach or cleaning agent...

 (H2O2). The released silver ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...

s are complexed subsequently by sodium thiosulfate
Sodium thiosulfate
Sodium thiosulfate , also spelled sodium thiosulphate, is a colorless crystalline compound that is more familiar as the pentahydrate, Na2S2O3•5H2O, an efflorescent, monoclinic crystalline substance also called sodium hyposulfite or “hypo.”...

.

Reduction and alkylation (r&a)

The staining and destaining is often followed by the reduction and alkylation (r&a) of the cystine
Cystine
Cystine is a dimeric amino acid formed by the oxidation of two cysteine residues that covalently link to make a disulfide bond. This organosulfur compound has the formula 2. It is a white solid, and melts at 247-249 °C...

s or cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...

s potentially embodied in the protein. Hereby the disulfide bond
Disulfide bond
In chemistry, a disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore R-S-S-R. The terminology is widely used in biochemistry...

s of the proteins are irreversibly broken up and the optimal unfolding of the tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...

 is obtained. The reduction to the thiol
Thiol
In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...

 is accomplished by the reaction with chemicals containing sulfhydryl or phosphine
Phosphine
Phosphine is the compound with the chemical formula PH3. It is a colorless, flammable, toxic gas. Pure phosphine is odourless, but technical grade samples have a highly unpleasant odor like garlic or rotting fish, due to the presence of substituted phosphine and diphosphine...

 groups such as dithiothreitol
Dithiothreitol
Dithiothreitol is the common name for a small-molecule redox reagent known as Cleland's reagent. DTT's formula is C4H10O2S2 and the molecular structure of its reduced form is shown at the right; its oxidized form is a disulfide-bonded 6-membered ring . Its name derives from the four-carbon...

 (DTT) or tris-2-carboxyethylphosphine hydrochloride (TCEP). In course of the subsequent irreversible alkylation of the SH groups with iodoacetamide
Iodoacetamide
2-Iodoacetamide is an alkylating agent used for peptide mapping purposes. Its actions are similar to those of iodoacetate. It is commonly used to bind covalently with the thiol group of cysteine so the protein cannot form disulfide bonds. Also used in ubiquitin studies as an inhibitor of...

 the cysteines are transformed to the stable S-carboxyamidomethylcysteine (CAM; adduct: -CH2-CONH2). The specific mass of the aminoacid cysteine is thereby increased from 103.01 Da
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...

 to 160.03 Da.

This chemical modification allows for proteins with a high number of disulfide bonds the successful identification as well as the highest peptide yield and sequence coverage. Due to the rareness of the aminoacid cysteine for most of the proteins the step of r&a does not effect any improvement of the mass spectrometric analysis. For the quantitative
Quantitative property
A quantitative property is one that exists in a range of magnitudes, and can therefore be measured with a number. Measurements of any particular quantitative property are expressed as a specific quantity, referred to as a unit, multiplied by a number. Examples of physical quantities are distance,...

 and homogeneous alkylation of cysteines the point of time for the modification is crucial. With denaturing electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...

 it is strongly recommended to perform the reaction before the execution of the electrophoresis, since there are free acrylamide
Acrylamide
Acrylamide is a chemical compound with the chemical formula C3H5NO. Its IUPAC name is prop-2-enamide. It is a white odourless crystalline solid, soluble in water, ethanol, ether, and chloroform. Acrylamide is incompatible with acids, bases, oxidizing agents, iron, and iron salts...

 monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...

s in the gel able to modify cysteines. The resulting acrylamide adducts are bound irreversible to the cysteines and can not be removed by subsequent r&a. The specific mass of the adduct is 174.05 Da
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...

.

In-gel digestion

Afterwards the eponymous step of the method is performed, the in-gel digestion of the proteins. By this procedure, the protein is cut enzymatically
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

 into a limited number of shorter fragments. These fragments are called peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...

s and allow for the identification of the protein with their characteristic mass and pattern. The serine protease
Serine protease
Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site.They are found ubiquitously in both eukaryotes and prokaryotes...

 trypsin
Trypsin
Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when...

 is the most common enzyme used in protein analytics. Trypsin cuts the peptide bond
Peptide bond
This article is about the peptide link found within biological molecules, such as proteins. A similar article for synthetic molecules is being created...

 specifically at the carboxyl end of the basic aminoacids arginine
Arginine
Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...

 and lysine
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....

. If there is an acidic amino acid like aspartic acid
Aspartic acid
Aspartic acid is an α-amino acid with the chemical formula HOOCCHCH2COOH. The carboxylate anion, salt, or ester of aspartic acid is known as aspartate. The L-isomer of aspartate is one of the 20 proteinogenic amino acids, i.e., the building blocks of proteins...

 or glutamic acid
Glutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...

 in direct neighborhood to the cutting site, the rate of hydrolysis is diminished, a proline
Proline
Proline is an α-amino acid, one of the twenty DNA-encoded amino acids. Its codons are CCU, CCC, CCA, and CCG. It is not an essential amino acid, which means that the human body can synthesize it. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary...

 C-terminal to the cutting site inhibits the hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...

 completely.

An undesirable side effect of the use of proteolytic enzymes is the self digestion of the protease. To avoid this, in the past Ca
Calcium
Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...

2+-ions were added to the digestion buffer. Nowadays most suppliers offer modified trypsin where selective methylation
Methylation
In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...

 of the lysines limits the autolytic activity to the arginine cutting sites. Unmodified trypsin has its highest activity between 35°C and 45°C. After the modification, the optimal temperature is changed to the range of 50°C to 55°C. Other enzymes used for in-gel digestion are the endoprotease
Protease
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....

s Lys-C, Glu-C, Asp-N  and Lys-N
Lys-N
Lys-N is a metalloendopeptidase found in the Japanese mushroom Grifola frondosa that cleaves proteins on the amino side of lysine residues. Lys-N is becoming a popular protease used for protein digestion in proteomics experiments...

. These proteases cut specifically at only one amino acid e.g. Asp-N cuts n-terminal of aspartic acid. Therefore a lower number of longer peptides is obtained.

The analysis of the complete primary sequence
Sequence
In mathematics, a sequence is an ordered list of objects . Like a set, it contains members , and the number of terms is called the length of the sequence. Unlike a set, order matters, and exactly the same elements can appear multiple times at different positions in the sequence...

 of a protein using only one protease is usually not possible. In those cases the digestion of the target protein in several approaches with different enzymes is recommended. The resulting overlapping peptides permit the assembly of the complete sequence of the protein.

For the digestion the proteins fixed in the matrix of the gel have to be made accessible for the protease. The permeation of the enzyme to the gel is believed to be facilitated by the dehydration
Dehydration
In physiology and medicine, dehydration is defined as the excessive loss of body fluid. It is literally the removal of water from an object; however, in physiological terms, it entails a deficiency of fluid within an organism...

 of the gel pieces by treatment with acetonitrile
Acetonitrile
Acetonitrile is the chemical compound with formula . This colourless liquid is the simplest organic nitrile. It is produced mainly as a byproduct of acrylonitrile manufacture...

 and subsequent swelling in the digestion buffer containing the protease. This procedure relies on the presumption that the protease permeates to the gel by the process of swelling. Different studies about the penetration of the enzymes to the gel showed the process to be almost completely driven by diffusion. The drying of the gel does not seem to support the process. Therefore, the improvement of the in-gel digestion has to be achieved by the reduction of the way of the enzyme to its substrate e.g. by cutting the gel to pieces as small as possible.

Usually, the in-gel digestion is run as an overnight process. For the use of trypsin as protease and a temperature of 37°C the time of incubation found in most protocols is 12-15 h. However, experiments about the duration of the digestion process showed that after 3 h there is enough material for successful mass spectrometric analysis. Furthermore, the optimisation of the conditions for the protease in temperature and pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

 allows for the completion of the digestion of a sample in 30 min.

Surfactant
Surfactant
Surfactants are compounds that lower the surface tension of a liquid, the interfacial tension between two liquids, or that between a liquid and a solid...

 (detergents) can aid in the solubilization and denaturing of proteins in the gel and thereby shorten digestion times and increase protein cleavage and the number and amount of extracted peptides, especially for lipophilic
Lipophilic
Lipophilicity, , refers to the ability of a chemical compound to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene. These non-polar solvents are themselves lipophilic — the axiom that like dissolves like generally holds true...

 proteins such as membrane protein
Membrane protein
A membrane protein is a protein molecule that is attached to, or associated with the membrane of a cell or an organelle. More than half of all proteins interact with membranes.-Function:...

s. Cleavable detergent
Cleavable detergent
Cleavable detergents, also known as cleavable surfactants, are special surfactants that are used in biochemistry and especially in proteomics to enhance protein denaturation and solubility...

s are detergents that are cleaved after digestion, often under acidic conditions. This makes the addition of detergents compatible with mass spectrometry.

Extraction

After finishing the digestion the peptides generated in this process have to be extracted from the gel matrix. This is accomplished by one or several extraction
Extraction (chemistry)
Extraction in chemistry is a separation process consisting in the separation of a substance from a matrix. It may refer to Liquid-liquid extraction, and Solid phase extraction....

 steps. The gel particles are incubated with an extraction solution and the supernatant is collected. In the first extraction, almost all of the peptide is recovered, the repetition of the extraction step can increase the yield of the whole process by only 5-10%. To meet the requirements of peptides with different physical and chemical properties an iterative extraction with basic or acidic solutions is performed. For the extraction of acidic peptides a solution similar to the concentration and composition of the digestion buffer is used; basic peptides are extracted in dependence to the intended mass spectrometric method with a low concentrated acidic solution of formic acid
Formic acid
Formic acid is the simplest carboxylic acid. Its chemical formula is HCOOH or HCO2H. It is an important intermediate in chemical synthesis and occurs naturally, most notably in the venom of bee and ant stings. In fact, its name comes from the Latin word for ant, formica, referring to its early...

 for ESI
Electrospray ionization
Electrospray ionization is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized...

 and trifluoroacetic acid
Trifluoroacetic acid
Trifluoroacetic acid is the simplest stable perfluorinated carboxylic acid chemical compound, with the formula CF3CO2H. It is a strong carboxylic acid due to the influence of the electronegative trifluoromethyl group. TFA is almost 100,000-fold more acidic than acetic acid...

 for MALDI respectively. Studies on model proteins showed a recovery of approximately 70–80% of the expected peptide yield by extraction from the gel.
Many protocols contain an additional fraction of acetonitrile to the extraction solution which, in concentrations above 30% (v/v), is effective in reducing the adsorption
Adsorption
Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...

 of peptides to the surface of reaction tubes and pipette
Pipette
A pipette is a laboratory tool used to transport a measured volume of liquid.-Use and variations:Pipettes are commonly used in molecular biology, analytical chemistry as well as medical tests...

 tips. The liquid of the pooled extracts is evaporated in a centrifugal evaporator
Centrifugal evaporator
A centrifugal evaporator is a device used in chemical and biochemical laboratories for the efficient and gentle evaporation of solvents from many samples at the same time, and samples contained in microtitre plates. If only one sample required evaporation then a rotary evaporator is most often used...

. If the volatile
Volatility (chemistry)
In chemistry and physics, volatility is the tendency of a substance to vaporize. Volatility is directly related to a substance's vapor pressure. At a given temperature, a substance with higher vapor pressure vaporizes more readily than a substance with a lower vapor pressure.The term is primarily...

 salt ammonium bicarbonate
Ammonium bicarbonate
Ammonium bicarbonate, a compound with formulaNH4, also called bicarbonate of ammonia, ammonium hydrogen carbonate, hartshorn, AmBic or powdered baking ammonia, is the bicarbonate salt of ammonia....

 was used for the basic extraction, it is partially removed in the drying process. The dried peptides can be stored at -20°C for at least six months.

Critical considerations and actual trends

Some major drawbacks of the common protocols for the in-gel digestion are the extended time need and the multiple processing steps making the method error-prone in respect to contamination
Contamination
Contamination is the presence of a minor and unwanted constituent in material, physical body, natural environment, at a workplace, etc.-Specifics:"Contamination" also has more specific meanings in science:...

s (especially keratin
Keratin
Keratin refers to a family of fibrous structural proteins. Keratin is the key of structural material making up the outer layer of human skin. It is also the key structural component of hair and nails...

). These disadvantages were largely removed by the development of optimised protocols and specialised reaction tubes.

More severe than the difficulties with handling are losses of material while processing the samples. The mass spectrometric protein analysis is often performed at the limit of detection, so even small losses can decide about success or failure of the whole analysis. These losses are due to washout during different processing steps, adsorption
Adsorption
Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...

 to the surface of reaction tubes and pipette
Pipette
A pipette is a laboratory tool used to transport a measured volume of liquid.-Use and variations:Pipettes are commonly used in molecular biology, analytical chemistry as well as medical tests...

 tips, incomplete extraction of peptides from the gel and/or bad ionisation of single peptides in the mass spectrometer. In dependence of the different physicochemical properties of the peptides the losses can vary between 15 and 50%. Due to this heterogeneity of the peptides up to now a universally valid solution for this major drawback of the method has not been found.

Commercial implementations

The commercial implementations of in-gel digestion have to be divided into products for high and for low throughput laboratories.

High-throughput

Due to the highly time consuming and work intensive standard procedure the method of in-gel digestion was limited to a relatively small number of protein spots to be processed at a time. Therefore it has been found to be the ideal object for automation
Automation
Automation is the use of control systems and information technologies to reduce the need for human work in the production of goods and services. In the scope of industrialization, automation is a step beyond mechanization...

 ambitions to overcome these limitations for industrial and service laboratories. Today, in laboratories where in-gel digestion is performed with a high-throughput the procedure is usually automated. The degree of automation varies from simple pipetting robot
Robot
A robot is a mechanical or virtual intelligent agent that can perform tasks automatically or with guidance, typically by remote control. In practice a robot is usually an electro-mechanical machine that is guided by computer and electronic programming. Robots can be autonomous, semi-autonomous or...

s to highly sophisticated all-in-one solutions, offering an automated workflow from gel to mass spectrometry. The systems usually consist of a spot picker, a digestion robot, and a spotter.

The spot picker is programmed with a picking list generated by an gel image analysis software
Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins...

 and excises the desired protein spots from a 2D gel
Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins...

. Manufacturers of spot picker are Herolab (Spothunter). The gel plugs are transferred to a microplate where the digestion robot performs the necessary steps for the in-gel digestion. Afterwards the spotter deposits the peptide solution on MALDI target plates or on microplates for automated ESI
Electrospray ionization
Electrospray ionization is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized...

-MS measurement.
Manufacturers of automated in-gel digestion systems are INTAVIS (DigestPro MSi), GE Healthcare
GE Healthcare
GE Healthcare is a division of GE Technology Infrastructure, which is itself a division of General Electric . It employs more than 46,000 people worldwide and is headquartered in Little Chalfont, Buckinghamshire, United Kingdom. GE Healthcare is the first GE business segment to be headquartered...

 (Ettan Series), Bruker Daltonics (PROTEINEER), Perkin Elmer
Applied Biosystems
Applied Biosystems, Inc. started as GeneCo , was the name of a pioneer biotechnology company founded in 1981 in Foster City, California, in the San Francisco Bay Area...

 (MultiPROBE II), and Shimadzu (Xcise).
The advantages of the automation other than the larger number of spots to be processed at a time are the reduced manual work and the improved standardisation. Due to the many handling steps of the method, the results of the manual process could vary depending on the dexterity of the user and the risk of contamination is high. Therefore, the quality of the results is described to be one main advantage of the automated process.

Drawbacks of automated solutions are the costs for robots, maintenance and consumables as well as the complicated setup of the process. Since the automated picking needs digitised information of the spot location, the analysis of the gel image for relevant spots has to be done by software requiring standardised imaging methods and special scanners. This lengthy procedure prevents the researcher from spontaneous identifications of a few interesting spots from a single gel as well as the need to operate the systems at full capacity. The resulting amount of data from the subsequent automated MS analysis is another problem of high throughput systems as their quality is often questionable and the evaluation of these data takes significantly longer than the collection.

Low-throughput

The mentioned drawbacks limit the reasonable use of automated in-gel digestion systems to the routine laboratory whereas the research laboratory with a demand to make a flexible use of the instruments of protein identification more often stays with the manual, low-throughput methods for in-gel digestion and MS analysis. This group of customers is targeted by the industry with several kit systems for in-gel digestion.

Most of the kit systems are mere collections of the chemicals and enzymes needed for the in-gel digestion whereas the underlying protocol remains unchanged from the manual standard procedure described above. The advantage of these products for the unexperienced customer lies in the guaranteed functioning of the diverse solutions in combination with a ready-made protocol for the process.

A few companies have tried to improve the handling process of in-gel digestion to allow even with manual sample preparation an easier and more standardised workflow. The MontageTM In-Gel Digest Kit from Millipore
Millipore Corporation
EMD Millipore, also known as Merck Millipore outside the United States and Canada, was founded in 1954, listed among the S&P 500 since the early 1990s, is an international biosciences company, known widely for its micrometer pore-size filters and tests...

 is based on the standard protocol, but enables processing of a large number of parallel samples by transferring the handling of the gel pieces to a modified 96 well microplate. The solutions for the diverse steps of in-gel digestion are pipetted into the wells of this plate whereas the removal of liquids is performed through the bottom of the wells by a vacuum pump
Vacuum pump
A vacuum pump is a device that removes gas molecules from a sealed volume in order to leave behind a partial vacuum. The first vacuum pump was invented in 1650 by Otto von Guericke.- Types :Pumps can be broadly categorized according to three techniques:...

. This system simplifies the handling of the multiple pipetting steps by the use of multichannel pipette
Pipette
A pipette is a laboratory tool used to transport a measured volume of liquid.-Use and variations:Pipettes are commonly used in molecular biology, analytical chemistry as well as medical tests...

s and even pipetting robots. Actually, some manufacturers of high-throughput systems have adopted the system to work with their robots. This illustrates the orientation of this kit solution to laboratories with a larger number of samples.

A completely different approach is made by the German company OMX GmbH. Their product line OMX-S is designed for the small scale use of up to 24 samples at a time by using a shortened protocol and specialised reaction tubes. The product is based on a critical evaluation of the conventional method of in-gel digestion where 30 steps and about 16 hours are needed for the processing from gel spot to peptide solution. The resulting protocol is reduced to four steps and about 2 hours process time by omitting steps not resulting in significant improvement of peptide yield and shortening the incubation time for digestion by increasing the temperature. The reaction tubes provided with this system enable the user to perform all steps from picking the spot to elution of peptides in one device. The gel spot is centrifuge
Centrifuge
A centrifuge is a piece of equipment, generally driven by an electric motor , that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis...

d to the reaction chamber through a small opening, a process by which the gel is torn to small pieces. The gel stays for the whole procedure in this reaction chamber, only the different buffers are added by manual pipetting and centrifugation and removed by centrifugation. The handling of in-gel digestion is significantly simplified and acclerated with this system, as well as the risks of contamination and loss of material are reduced. However, the processing of a larger number of samples is due to the manual processing of every single spot still work intensive and not comparable to automated systems.

External links

  • Flash film illustrating the experimental procedure of the optimised in-gel digestion as described in Granvogl et al.

See also

  • Zymography
    Zymography
    Zymography is an electrophoretic technique, based on SDS-PAGE, that includes a substrate co-polymerized with the polyacrylamide gel, for the detection of enzyme activity.-Procedure:...

    , an unrelated technique in molecular biology which also involves the digestion of proteins in an electrophoretic gel
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