The in-gel digestion step is a part of the sample preparation for the mass spectrometric identification of

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

s in course of proteomic analysis. The method was introduced in 1992 by Rosenfeld.Rosenfeld, J et al., ''Anal Biochem'', 1992, 203 (1), 173-9. Innumerable modifications and improvements in the basic elements of the procedure remain.Hellman, U et al., ''Anal Biochem'', 1995, 224 (1), 451-455.Jeno, P et al., ''Anal Biochem'', 1995, 224 (1), 75-82.Shevchenko, A et al., ''Anal Chem'', 1996, 68 (5), 850-8.Borchers, C et al., ''Anal Chem'', 2000, 72 (6), 1163-8.Shevchenko, A et al., ''Nat Protoc'', 2006, 1 (6), 2856-60.Granvogl, B et al., ''Proteomics'', 2007, 7 (5), 642-54. The in-gel digestion step primarily comprises the four steps; destaining, reduction and

alkylation Alkylation is a chemical reaction that entails transfer of an alkyl group. The alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion, or a carbene (or their equivalents). Alkylating agents are reagents for effecting al ...

(R&A) of the

cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...

s in the protein, proteolytic cleavage of the protein and extraction of the generated

peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...

Destaining

Proteins which were separated by 1D or 2D

PAGE Page most commonly refers to: * Page (paper), one side of a leaf of paper, as in a book Page, PAGE, pages, or paging may also refer to: Roles * Page (assistance occupation), a professional occupation * Page (servant), traditionally a young m ...

are usually visualised by staining with

dye Juan de Guillebon, better known by his stage name DyE, is a French musician. He is known for the music video of the single "Fantasy Fantasy is a genre of speculative fiction that involves supernatural or Magic (supernatural), magical ele ...

s like Coomassie brilliant blue (CBB) or

silver Silver is a chemical element; it has Symbol (chemistry), symbol Ag () and atomic number 47. A soft, whitish-gray, lustrous transition metal, it exhibits the highest electrical conductivity, thermal conductivity, and reflectivity of any metal. ...

. 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

s before proceeding. The destaining solution for CBB contains usually the buffer

salt In common usage, salt is a mineral composed primarily of sodium chloride (NaCl). When used in food, especially in granulated form, it is more formally called table salt. In the form of a natural crystalline mineral, salt is also known as r ...

ammonium bicarbonate (NH₄HCO₃) and a fraction of 30%-50% organic

solvent A solvent (from the Latin language, Latin ''wikt:solvo#Latin, solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a Solution (chemistry), solution. A solvent is usually a liquid but can also be a solid, a gas ...

(mostly

acetonitrile Acetonitrile, often abbreviated MeCN (methyl cyanide), is the chemical compound with the formula and structure . This colourless liquid is the simplest organic nitrile (hydrogen cyanide is a simpler nitrile, but the cyanide anion is not class ...

). The hydrophobic interactions between protein and CBB are reduced by the organic fraction of the solution.Jin, Y and Manabe, T, ''Electrophoresis'', 2005, 26 (6), 1019-28. At the same time, the ionic part of the solution diminishes the

electrostatic Electrostatics is a branch of physics that studies slow-moving or stationary electric charges. Since classical times, it has been known that some materials, such as amber, attract lightweight particles after rubbing. The Greek word (), mean ...

bonds between the

and the positively charged

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

s of the protein. In contrast to a mixture of

water Water is an inorganic compound with the chemical formula . It is a transparent, tasteless, odorless, and Color of water, nearly colorless chemical substance. It is the main constituent of Earth's hydrosphere and the fluids of all known liv ...

with organic solvent the effectivity of destaining is increased. An increase of

temperature Temperature is a physical quantity that quantitatively expresses the attribute of hotness or coldness. Temperature is measurement, measured with a thermometer. It reflects the average kinetic energy of the vibrating and colliding atoms making ...

promotes the destaining process.Lloyd, MD, ''Anal Biochem'', 1996, 241 (1), 139-40. To a certain degree (< 10%) the destaining procedure is accompanied with a loss of protein.Speicher, KD et al., ''Journal of Biomolecular Techniques'', 2000, 11 (2), 74-86. Furthermore, the removal of CBB does not affect the yield of

s in the mass spectrometric measurement.Terry, DE et al., ''J Am Soc Mass Spectrom'', 2004, 15 (6), 784-94. In the case of silver stained protein bands the destaining is accomplished by

oxidation Redox ( , , reduction–oxidation or oxidation–reduction) is a type of chemical reaction in which the oxidation states of the reactants change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is ...

of the metallic

attached to the protein by potassium ferricyanide or

hydrogen peroxide Hydrogen peroxide is a chemical compound with the formula . In its pure form, it is a very pale blue liquid that is slightly more viscosity, viscous than Properties of water, water. It is used as an oxidizer, bleaching agent, and antiseptic, usua ...

(H₂O₂).Gharahdaghi, F et al., ''Electrophoresis'', 1999, 20 (3), 601-5.Sumner, LW et al., ''Rapid Commun Mass Spectrom'', 2002, 16 (3), 160-8. The released silver ions are complexed subsequently by sodium thiosulfate.

Reduction and alkylation (R & A)

The staining and destaining of gels is often followed by the reduction and alkylation (r&a) of the cystines or

s in the proteins. Hereby, the disulfide bonds of the proteins are irreversibly broken up and the optimal unfolding of the

tertiary structure Protein tertiary structure is the three-dimensional shape of a protein. The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains. Amino acid side chains and the ...

is obtained. The reduction to the thiol is accomplished by the reaction with chemicals containing

sulfhydryl In organic chemistry, a thiol (; ), or thiol derivative, is any organosulfur compound of the form , where R represents an alkyl or other organic substituent. The functional group itself is referred to as either a thiol group or a sulfhydryl grou ...

phosphine Phosphine (IUPAC name: phosphane) is a colorless, flammable, highly toxic compound with the chemical formula , classed as a pnictogen hydride. Pure phosphine is odorless, but technical grade samples have a highly unpleasant odor like rotting ...

groups such as dithiothreitol (DTT) or tris-2-carboxyethylphosphine hydrochloride (TCEP). In course of the subsequent irreversible alkylation of the SH groups with iodoacetamide the cysteines are transformed to the stable S-carboxyamidomethylcysteine (CAM; adduct: -CH₂-CONH₂). The molecular weight of the cysteine amino-acid residue is thereby increased from 103.01 Da to 160.03 Da. Reduction and alkylation of cysteine residues improves peptide yield and sequence coverage and the identification of proteins with a high number of disulfide bonds.Hale, JE et al., ''Anal Biochem'', 2004, 333 (1), 174-81.Katayama, H et al., ''Rapid Commun Mass Spectrom'', 2004, 18 (20), 2388-94. Due to the rareness of the amino acid cysteine for most of the proteins the step of r&a does not effect any improvement of the mass spectrometric analysis.Havlis, J et al., ''Anal Chem'', 2003, 75 (6), 1300-6.Shevchenko, A and Shevchenko, A, ''Anal Biochem'', 2001, 296 (2), 279-83. For the quantitative and homogeneous alkylation of cysteines the position of the modification step in the sample-preparation process is crucial. With denaturing electrophoresis it is strongly recommended to perform the reaction before the execution of the electrophoresis, since there are free acrylamide

monomer A monomer ( ; ''mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or two- or three-dimensional network in a process called polymerization. Classification Chemis ...

s in the gel able to modify cysteine residues irreversibly.Hamdan, M et al., ''Electrophoresis'', 2001, 22 (9), 1633-44.Mineki, R et al., ''Proteomics'', 2002, 2 (12), 1672-81.Sechi, S and Chait, BT, ''Anal Chem'', 1998, 70 (24), 5150-8.Herbert, B et al., ''Electrophoresis'', 2001, 22 (10), 2046-57. The resulting acrylamide adducts have a molecular weight of 174.05 Da.

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 into a limited number of shorter fragments. These fragments are called

s and allow for the identification of the protein with their characteristic mass and pattern. The

serine protease Serine proteases (or serine endopeptidases) are enzymes that cleave peptide bonds in proteins. Serine serves as the nucleophilic amino acid at the (enzyme's) active site. They are found ubiquitously in both eukaryotes and prokaryotes. Serin ...

trypsin Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting long chains of amino acids into smaller pieces. It is a serine protease from the PA clan superfamily, found in the dig ...

is the most common enzyme used in protein analytics. Trypsin cuts the

peptide bond In organic chemistry, a peptide bond is an amide type of covalent chemical bond linking two consecutive alpha-amino acids from C1 (carbon number one) of one alpha-amino acid and N2 (nitrogen number two) of another, along a peptide or protein cha ...

specifically at the carboxyl end of the basic aminoacids

arginine Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidinium, guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) a ...

and

lysine Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. Lysine contains an α-amino group (which is in the protonated form when the lysine is dissolved in water at physiological pH), an α-carboxylic acid group ( ...

. If there is an acidic amino acid like

aspartic acid Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protei ...

glutamic acid Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α- amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can ...

in direct neighborhood to the cutting site, the rate of hydrolysis is diminished, a proline C-terminal to the cutting site inhibits the

hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...

completely.Thiede, B et al., ''Rapid Commun Mass Spectrom'', 2000, 14 (6), 496-502. An undesirable side effect of the use of proteolytic enzymes is the self digestion of the protease. To avoid this, in the past Ca²⁺-ions were added to the digestion buffer.Vajda, T and Garai, A, ''J Inorg Biochem'', 1981, 15 (4), 307-15.Sipos, T and Merkel, JR, ''Biochemistry'', 1970, 9 (14), 2766-75. Nowadays most suppliers offer modified trypsin where selective

methylation Methylation, in the chemistry, chemical sciences, is the addition of a methyl group on a substrate (chemistry), substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replac ...

of the lysines limits the autolytic activity to the arginine cutting sites.Rice, RH et al., ''Biochimica et Biophysica Acta'', 1977, 492 (2), 316-321. 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.Finehout, EJ et al., ''Proteomics'', 2005, 5 (9), 2319-21. Other enzymes used for in-gel digestion are the endo

protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalysis, catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products ...

s Lys-C,Michalski, WP and Shiell, BJ, ''Analytica Chimica Acta '', 1999, 383 (1-2), 27-46.Jekel, PA et al., ''Anal Biochem'', 1983, 134 (2), 347-54.Patterson, SD, ''Electrophoresis'', 1995, 16 (7), 1104-14. Glu-C,Scheler, C et al., ''Electrophoresis'', 1998, 19 (6), 918-27.Houmard, J and Drapeau, GR, ''Proc Natl Acad Sci U S A'', 1972, 69 (12), 3506-9.Farah, MA et al., ''Biochim Biophys Acta'', 2005, 1725 (3), 269-82. Asp-NWang, L et al., ''Pharm Res'', 2005, 22 (8), 1338-49. and Lys-N. 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 In mathematics, a sequence is an enumerated collection of objects in which repetitions are allowed and order matters. Like a set, it contains members (also called ''elements'', or ''terms''). The number of elements (possibly infinite) is cal ...

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.Choudhary, G et al., ''J Proteome Res'', 2003, 2 (1), 59-67.Wa, C et al., ''Anal Biochem'', 2006, 349 (2), 229-41. 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 In physiology, dehydration is a lack of total body water that disrupts metabolic processes. It occurs when free water loss exceeds intake, often resulting from excessive sweating, health conditions, or inadequate consumption of water. Mild deh ...

of the gel pieces by treatment with

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.Finehout, EJ and Lee, KH, ''Electrophoresis'', 2003, 24 (19-20), 3508-16. Furthermore, the optimisation of the conditions for the protease in temperature and pH allows for the completion of the digestion of a sample in 30 min.

Surfactant Surfactants are chemical compounds that decrease the surface tension or interfacial tension between two liquids, a liquid and a gas, or a liquid and a solid. The word ''surfactant'' is a Blend word, blend of "surface-active agent", coined in ...

(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 Lipophilicity (from Greek language, Greek λίπος "fat" and :wikt:φίλος, φίλος "friendly") is the ability of a chemical compound to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene. Such compounds are c ...

proteins such as membrane proteins. Cleavable detergents 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 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 (), systematically named methanoic acid, is the simplest carboxylic acid. It has the chemical formula HCOOH and structure . This acid is an important intermediate in chemical synthesis and occurs naturally, most notably in some an ...

for ESI and

trifluoroacetic acid Trifluoroacetic acid (TFA) is a synthetic organofluorine compound with the chemical formula CF3CO2H. It belongs to the subclass of per- and polyfluoroalkyl substances (PFASs) known as ultrashort-chain perfluoroalkyl acids (PFAAs). TFA is not ...

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 is the adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid to a surface. This process creates a film of the ''adsorbate'' on the surface of the ''adsorbent''. This process differs from absorption, in which a ...

of peptides to the surface of reaction tubes and pipette tips.Erdjument-Bromage, H et al., ''J Chromatogr A'', 1998, 826 (2), 167-81. The liquid of the pooled extracts is evaporated in a centrifugal evaporator. If the volatile salt ammonium bicarbonate 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 needed and the multiple processing steps, making the method error-prone with respect to

contamination Contamination is the presence of a constituent, impurity, or some other undesirable element that renders something unsuitable, unfit or harmful for the physical body, natural environment, workplace, etc. Types of contamination Within the scien ...

s (especially

keratin Keratin () is one of a family of structural fibrous proteins also known as ''scleroproteins''. It is the key structural material making up Scale (anatomy), scales, hair, Nail (anatomy), nails, feathers, horn (anatomy), horns, claws, Hoof, hoove ...

). 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 dictate success or failure of the whole analysis. These losses are due to washout during different processing steps,

to the surface of reaction tubes and pipette tips, incomplete extraction of peptides from the gel and/or bad

ionisation Ionization or ionisation is the process by which an atom or a molecule acquires a negative or positive charge by gaining or losing electrons, often in conjunction with other chemical changes. The resulting electrically charged atom or molecule i ...

of single peptides in the

mass spectrometer Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a '' mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is us ...

.Stewart, II et al., ''Rapid Commun Mass Spectrom'', 2001, 15 (24), 2456-65. Depending on the physicochemical properties of the peptides, losses can vary between 15 and 50%. Due to the inherent 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 describes a wide range of technologies that reduce human intervention in processes, mainly by predetermining decision criteria, subprocess relationships, and related actions, as well as embodying those predeterminations in machine ...

ambitions to overcome these limitations for industrial and service laboratories.Houthaeve, T et al., ''Journal of Protein Chemistry'', 1997, 16 (5), 343-348. Today, in laboratories where in-gel digestion is performed in high-throughput quantities, the procedure is usually automated. The degree of automation varies from simple pipetting

robot A robot is a machine—especially one Computer program, programmable by a computer—capable of carrying out a complex series of actions Automation, automatically. A robot can be guided by an external control device, or the robot control, co ...

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 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.Canelle, L et al., ''Rapid Communications in Mass Spectrometry'', 2004, 18 (23), 2785–2794. 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.Stead, DA et al., ''Brief Bioinform'', 2008Hu, J et al., ''Brief Funct Genomic Proteomic'', 2005, 3 (4), 322-31.

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 inexperienced 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 Montage In-Gel Digest Kit from Millipore 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 A vacuum pump is a type of pump device that draws gas particles from a sealed volume in order to leave behind a partial vacuum. The first vacuum pump was invented in 1650 by Otto von Guericke, and was preceded by the suction pump, which dates to ...

. This system simplifies the handling of the multiple pipetting steps by the use of multichannel pipettes 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.

References

External links

Flash film
illustrating the experimental procedure of the optimised in-gel digestion as described in Granvogl et al. {{DEFAULTSORT:In-Gel Digestion Proteins Mass spectrometry