In-gel digestion

In- gel digestion is part of the sample preparation for mass spectrometry analysis of proteins in the context of proteome analysis. In- gel digestion mainly comprises the four steps of bleaching, reduction and alkylation (R & A) of the cysteines contained in the proteins, proteolytic cleavage of the proteins and the extraction of the peptides produced.

The method was in 1992 by Rosenfeld et al. introduced. Despite numerous changes and improvements to the process, the basic ingredients have until today largely retained.

• 3.1 High-Throughput
• 3.2 low throughput

Principle

Decolorization

After cutting of the separated by 1D or 2D PAGE and dyes ( for example, Coomassie brilliant blue (CBB ) or silver ) protein bands visualized or spots there is a discoloration of the proteins. The use of Coomassie for visualization of proteins prepares the fewest problems in the subsequent mass spectrometric analysis. Therefore, the method is widely used despite its lower sensitivity.

The destaining solution for CBB usually contains the buffer - ammonium salt ( NH4HCO3 ) and an amount of 30-50 % organic solvent (usually acetonitrile). The hydrophobic interactions between the protein and Coomassie dye to be reduced in the organic solvent. At the same time the amount of salt reduces the electrostatic interaction between the dye molecules and the positively charged amino acids of the proteins. In comparison with a mixture of water and organic solvent, the effectiveness of decolorization is improved. An increase in temperature promotes the deinking process. The discoloration is usually associated with a loss of less than ten percent of the protein .. In addition, the removal of the dye generally not improved the peptide yield.

In the case of silver- stained bands of the decolorization is carried out by oxidation of the deposited metallic silver to the proteins using Kaliumhexacyanidoferrat (III) or hydrogen peroxide ( H2O2). The released heavy metal ions are subsequently complexed with sodium thiosulfate.

Reduction and alkylation

At the staining and discoloration of the proteins, a reduction and alkylation of cystine and cysteine ​​residues potentially contained is often connected. The disulfide bonds of the protein to be separated and therefore achieves an optimal development of the protein. Reduction to thiol is accomplished by reaction with chemicals containing sulfhydryl or phosphine (for example dithiothreitol ( DTT) or tris-2- carboxyethylphosphine hydrochloride ( TCEP ) ). Transferred :; ( -CH2- CONH2 adduct CAM) During subsequent irreversible alkylation of the SH groups with iodoacetamide the cysteines are in the stable S- Carboxyamidomethylcystein. Thus, the specific mass of the amino acid cysteine ​​increased from 103.01 to 160.03 Da.

By the modification of proteins is ensured with a large number of disulfide bonds, the identification peptide and a maximum yield and sequence coverage. Because of the relative rarity of the amino acid cysteine ​​brings this step a large part of the protein does not significantly improve. For a quantitative and homogeneous alkylation of the cysteines of the date of the modification is crucial. In denaturing electrophoresis, it lends itself to the reaction before the separation perform because free acrylamide monomers can also modify cysteines. The resulting acrylamide adducts are also irreversibly bound to the cysteines. The specific mass of the adduct is 174.05 Da.

In -gel digestion

Then, the namesake for the method step, the in-gel digestion of the protein occurs. In this case, the protein is enzymatically into a defined number of shorter fragments characteristic mass ( peptides) split that can be used in its identification. The serine protease trypsin is in the proteome most widely used protease. Trypsin cleaves peptide bonds at the carboxy end of the specific basic amino acids arginine and lysine. Located just adjacent to the interface, an acidic amino acid ( aspartate or glutamate ) the rate of hydrolysis is limited. No cleavage occurs at a C -terminal to the interface located proline.

As an unwanted side effect occurs with proteolytic digestion on the Eigenverdau the protease. To prevent this, the digestion buffer were used to Ca2 ions are added. Today, modified trypsin is offered by most manufacturers. Selective methylation of the lysines present in the trypsin Eigenverdau may be limited to its arginine- containing peptides. Unmodified trypsin has its highest activity between 35 and 45 ° C. After the modification, the optimum temperature shifts to 50 to 55 ° C. In addition to trypsin, among others find the endoproteases Lys -C, Glu -C and Asp -N use. These proteases cut specifically at only one amino acid. This yields a limited number of longer peptides.

The analysis of the complete primary sequence of a protein having only one protease is not usually possible. In this case, the target protein can be digested into a plurality of batches of different proteases. This partially overlapping peptides are generated, which can then be assembled to the overall sequence

For the digestion, the proteins in the gel, fixed for the protease to be made available. The gel pieces are to facilitate the penetration of the enzyme dehydratiert first with acetonitrile and then swollen in a protease-containing digestion buffer. The method is based on the assumption that the swelling process, the protease is added to the gel. However, the penetration of the protease into the gel matrix is a largely diffusion- dependent process. Drying of the gel therefore appears not to support the process of diffusion. For optimization of the in-gel digestion hence lends itself to a maximum comminution of the gel matrix in order to reduce the diffusion distance for the protease drastically.

In- gel digestion takes place normally overnight. When using trypsin as the protease at a temperature of 37 ° C, the incubation period is about 12-15 hours. However, experiments have shown that already after three hours is enough material is provided for the mass spectrometric analysis. The optimization of the conditions for the protease (pH, temperature) allowed the complete digestion of a sample but also in 30 minutes.

Extraction

After completion of the Verdauprozesses the resulting peptides have to be extracted from the gel matrix. This usually happens in two extraction steps. Here, the gel particles are incubated in an extraction solution and lifted the dissolved out of the gel particles peptides with the supernatant. The principal amount of peptide is already obtained with an extraction step. Additional extractions improve peptide yield, taken together, only about five to ten percent. To ensure the extraction of peptides with different physico-chemical properties, serial extractions are carried out in alkaline or acid solutions. For the extraction of the acidic peptides finds an analog in concentration and composition of the digestion buffer solution using; The basic peptides are extracted depending on the type of MS analysis, either with formic acid (ESI) or with trifluoroacetic acid ( MALDI) in low concentration. Based on model proteins, it was confirmed that about 70-80 % of the expected amount of peptide can be extracted from the gel. Many protocols for the extraction additionally comprise a proportion of acetonitrile is reduced from a concentration of 30 % (v / v), the adsorption of peptides to the surfaces of reaction vessels and pipette tips. The combined extracts are then evaporated in a vacuum centrifuge to dryness. Is the volatile ammonium bicarbonate used as the buffer salt to the alkaline extraction, as this will be removed in part during the drying process. In the dried form, the isolated peptides can be stored at -20 ° C for at least six months.

Problems

Important disadvantages of the methods for in-gel digestion, the susceptibility to contamination (by a keratin) and the high time requirement due to the multiple processing steps. These disadvantages could be mitigated by the development of optimized protocols and specialized reaction vessels.

Serious are the losses of sample material during preparation, which can decide in the often operating at the limit of detection mass spectrometric protein analysis of the success of the method. These occur, for example, by washing during the individual processing steps, adsorption to surfaces of reaction vessels or pipette tips, incomplete extraction of the peptide from the gel and / or due to poor ionization of individual peptides. As a function of the physico- chemical properties of the peptides of the losses between 15 and 50 % may vary. These problems could be solved universal due to the heterogeneity of the various peptides with respect to these properties has not.

Applications

In the commercial realizations of the in-gel digestion, one can distinguish between solutions for low-and high throughput.

High-throughput

Due to the high time and labor required for the standard process of the manual in-gel digestion was limited to a relatively small number of samples could process a person in a given time. With the need for large-scale test series in proteomics to perform and provide the opportunities that modern mass spectrometric methods and the performance of computer technology in terms of the automatic measurement and evaluation, the development of an automated sample preparation has been initiated. Today, standard automated solutions are used in laboratories that need to process a large number of samples routinely. The level of automation ranges from simple pipetting robots to sophisticated proteomics workstations that automatically perform all steps from the gel to the mass spectrometer. These systems usually consist of a spot picker, digestion robot and a spotter.

The spot picker is programmed with a pick- list that is generated in a 2-D gel analysis program. According to this list the device the desired protein spots stands out from a 2D gel and transferred them to a microtiter plate. There the digestion robot performs the necessary steps for the in-gel digestion. The spotter generated eventually with the peptide solution, the spots on the MALDI target and feeds a microtiter plate suitable for automated ESI- MS measurement. Manufacturer of automatic Bankrupts In - gel digestion systems are Intavis ( DigestPro ), GE Healthcare ( Ettan Series), Bruker Daltonics ( PROTEINEER ), Perkin Elmer ( Multiprobe ® II) and Shimadzu ( Xcise ). In addition to the large number of samples to be processed at the same time the advantage of the automation is in the reduced personnel requirements and the enhanced standardization of the procedure. In the manual in-gel digestion, the results may be dependent on the experience of the user and there is a great risk of contamination due to the many steps to be performed. The improved earnings quality is therefore called as a major advantage of the fully automated processing of the samples.

Disadvantages of the automated solutions is the cost for robot maintenance and often proprietary consumables as well as the complicated operation of the system. So the automatic punching relies on digitized information of the spot position, which necessitates the use of gel analysis programs and special scanners. This lengthy procedure and the (economic ) need to operate the system only with a certain number of samples (the smallest customary for this microtiter plate holds 96 samples ) prevents the spontaneous selecting and analyzing a small number of spots from a single gel. Furthermore, to consider the amount of data generated from the following also automated MS analysis very critical, because their quality is often questionable and hence the need for careful evaluation, which takes much longer to charge than these data.

Low-throughput

The disadvantage mentioned limit the reasonable use of automated in-gel digestion systems on routine laboratories during research laboratories due to their need for a more flexible use of the instruments often remain for protein identification in the manual methods. For this group of users the industry kit systems developed for the in- gel digestion.

Most of these systems are mere kit collections of chemicals and enzymes that are needed for the in-gel digestion, wherein the implementation of the method is not changed as such. The advantage for the inexperienced clients is that the chemicals and enzymes and the Protocol are purchased from a manufacturer, making the functioning of the method is guaranteed. Manufacturers of these systems are Kit Sigma - Aldrich ( trypsin Profile IGD Kit), Pierce ( In - Gel Tryptic Digestion Kit) and Agilent (Protein In - gel Tryptic Digestion Kit).

Only a few companies have tried to improve the handling of the in-gel digestion in order to achieve a simpler and standardized workflow without robots. The MontageTM in-gel digest kit from Millipore based on the standard protocol for in-gel digestion, but postponed the process steps in a special microtiter plate, resulting in a larger number of samples can be processed simultaneously. The solutions for the various steps will be added with a pipette, whereas the suction takes place at the bottom by the bottom of the plate using a vacuum. This reduces the number of pipetting steps for the user is reduced because for a manual removal of the liquids can be dispensed with and on the other hand feeding using multichannel pipettes or even pipetting robots. Some manufacturers have even this system adapted for use with their automated high -throughput systems. The details of the process to illustrate the orientation of the Millipore system to laboratories have at least an average throughput.

A completely different approach has followed the German company OMX GmbH. The range OMX- S ® is focused on the simultaneous processing of up to 24 samples, with both a specially modified protocol as well as newly developed reaction vessels are used. The system is the result of a critical analysis of the conventional protocol for in-gel digestion, where about 30 steps and 16 hours for the entire process from gel to the peptide solution is estimated. The result of the study was a shortened four steps and about two hours protocol. Process steps that do not lead to a significant improvement of the final peptide - yield have been omitted therein, and reduces the necessary incubation time for digestion by increasing the temperature. The reaction vessels used in the system, special permit to carry out all the steps of the in-gel digestion, by punching to the peptide extraction, in a vessel. The gel spot is gouged in this process with the built- piercing tool and centrifuged into the reaction chamber, where it must pass through a small opening and is thereby torn into small pieces. In this reaction, the gel space for the entire process remains, the solutions are simply added through the piercing channel and removed by centrifugation again. This method provides a significant simplification and acceleration of the manual in-gel digestion is, but as each sample must be individually processed, the processing of larger approaches is hereby still does not compare very labor intensive and in this respect with the automated systems.