In-gel digestion

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

The method was developed in 1992 by Rosenfeld et al. introduced. Despite countless changes and improvements to the process, the basic components have largely been preserved to this day.

principle

Discoloration

After cutting out the protein bands or spots, separated by means of 1D or 2D PAGE and visualized with dyes (for example Coomassie brilliant blue (CBB) or silver ), the proteins are decolorized . The use of Coomassie for the visualization of proteins causes the fewest problems in the subsequent mass spectrometric analysis . This is why the method is widespread despite its lower sensitivity .

The destaining solution for CBB usually contains the buffer - salt ammonium bicarbonate (NH 4 HCO 3 ) and a proportion of 30-50% organic solvent (usually acetonitrile ). The hydrophobic interactions between protein and Coomassie dye are reduced in the organic solvent. At the same time, the salt content reduces the electrostatic interactions between the dye molecules and the positively charged amino acids of the proteins. In comparison with a mixture of water and organic solvent, the decolorization efficiency is improved. An increase in temperature favors the decolorization process. The discoloration is usually associated with a loss of less than ten percent of the protein. In addition, the removal of the dye does not fundamentally improve the peptide yield .

In the case of silver-colored bands, the discoloration takes place through the oxidation of the metallic silver attached to the proteins with the aid of potassium hexacyanidoferrate (III) or hydrogen peroxide (H 2 O 2 ). The released heavy metal ions are then complexed with sodium thiosulphate .

Reduction and alkylation

The coloring and discoloration of the proteins are often followed by a reduction and alkylation of the potentially contained cystines or cysteines . The disulphide bridges of the protein are separated and thus optimal development of the protein is achieved. The reduction of thiol is accomplished via reaction with chemicals that sulfhydryl or phosphine groups (for example, dithiothreitol (DTT) or tris-2-carboxyethyl phosphine hydrochloride contain (TCEP)). In the course of the subsequent irreversible alkylation of the SH groups with iodoacetamide , the cysteines are converted into the stable S-carboxyamidomethylcysteine ​​(CAM; adduct: -CH 2 -CONH 2 ). This increases the specific mass for the amino acid cysteine ​​from 103.01 to 160.03  Da .

The modification ensures identification and maximum peptide yield and sequence coverage for proteins with a high number of disulfide bridges . Because of the relative rarity of the amino acid cysteine, this step does not bring about any significant improvement for a large part of the proteins. The point in time of the modification is decisive for a quantitative and homogeneous alkylation of the cysteines. When denaturing electrophoresis it makes sense to carry out the reaction before the separation, since free acrylamide - monomers can modify also 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

This is followed by the eponymous step for the method, the in-gel digestion of the protein . The protein is enzymatically split into a defined number of shorter fragments with a characteristic mass (peptides) that can be used for its identification. The serine protease trypsin is the most widely used protease in proteome analysis . Trypsin cleaves peptide bonds specifically at the carboxy end of the basic amino acids arginine and lysine . If there is an acidic amino acid ( aspartate or glutamate ) immediately adjacent to the interface , the rate of hydrolysis is restricted. There is no cleavage in the case of a proline located C-terminally to the interface .

The self-digestion of the protease occurs as an undesirable side effect of proteolytic digestion. To prevent this, Ca 2+ ions were previously added to the digestion buffer . Today, most manufacturers offer modified trypsin. By selective methylation of the lysines contained in the trypsin, self-digestion can be restricted to its arginine-containing peptides. Unmodified trypsin has its highest activity between 35 and 45 ° C. After the modification, the temperature optimum shifts to 50 to 55 ° C. In addition to trypsin, the endoproteases Lys-C, Glu-C, and Asp-N are used. These proteases cut specifically on only one amino acid. This gives a limited number of longer peptides.

The analysis of the complete primary sequence of a protein with only one protease is usually not possible. In this case, the target protein can be digested in several batches with different proteases. In doing so, partially overlapping peptides are generated, which can then be combined to form the overall sequence

For digestion, the proteins fixed in the gel must be made accessible to the protease. In order to facilitate the penetration of the enzyme, the gel pieces are first dehydrated with acetonitrile and then swollen in a protease- containing digestion buffer . The method is based on the assumption that the protease is absorbed into the gel during the swelling process. The penetration of the protease into the gel matrix is, however, a largely diffusion-dependent process. The drying of the gel therefore does not seem to support the diffusion process. In order to optimize the in-gel digestion, maximum comminution of the gel matrix is ​​therefore advisable in order to drastically reduce the diffusion distance for the protease.

The in-gel digestion usually takes place overnight. When using trypsin as protease and at a temperature of 37 ° C, the incubation time is about 12–15 hours. Tests have shown, however, that enough material is available for mass spectrometric analysis after just three hours . Optimizing the conditions for the protease ( pH , temperature) allows a sample to be completely digested in 30 minutes.

extraction

After the digestion process has ended, the resulting peptides must be extracted from the gel matrix . This usually takes place in two extraction steps. The gel particles are incubated in an extraction solution and the peptides removed from the gel particles are lifted off with the supernatant. The majority of the peptide is obtained in just one extraction step. Additional extractions improve the total peptide yield by only about five to ten percent. In order to ensure the extraction of peptides with different physico-chemical properties, serial extractions are carried out in basic or acidic solutions. A solution analogous in concentration and composition to the digestion buffer is used for the extraction of the acidic peptides ; the basic peptides are extracted either with formic acid ( ESI ) or with trifluoroacetic acid ( MALDI ) in low concentration, depending on the type of MS analysis . Using model proteins , it was proven that around 70-80% of the expected amount of peptide is extracted from the gel. Many protocols also contain a proportion of acetonitrile for extraction , which from a concentration of 30% (v / v) reduces the adsorption of peptides on the surfaces of reaction vessels and pipette tips . The combined extracts are then evaporated to dryness in a vacuum centrifuge. If the volatile ammonium hydrogen carbonate was used as a buffer salt for the basic extraction, this is partly removed during the drying process. In the dried form, the isolated peptides can be stored at −20 ° C for at least six months.

Problems

Decisive disadvantages of the methods for in-gel digestion are the susceptibility to contamination (especially keratin ) and the high time required due to the multiple processing steps. These disadvantages could be reduced by the development of optimized protocols and specialized reaction vessels.

More serious are the losses of sample material during preparation, which can be decisive for the success of the method in the case of mass spectrometric protein analysis, which often operates at the detection limit. These occur, for example, as a result of washing out during the individual processing steps , adsorption on surfaces of reaction vessels or pipette tips , incomplete extraction of the peptides from the gel and / or poor ionization of individual peptides. Depending on the physico-chemical properties of the peptides, the losses can vary between 15 and 50%. Due to the heterogeneity of the different peptides with regard to these properties, these problems could not yet be solved in a generally applicable manner.

Applications

In the commercial implementations of in-gel digestion, a distinction can be made between solutions for low and high throughput.

High throughput

Due to the high time and effort required for the standard procedure, manual in-gel digestion was limited to a relatively small number of samples that a person could process in a given time. With the need to carry out large-scale test series in proteomics and the possibilities offered by modern mass spectrometric methods and the performance of computer technology with regard to automatic measurement and evaluation, the development of an automated sample preparation was initiated. Today, automated solutions are used as standard in laboratories that routinely process large numbers of samples. The degree of automation ranges from simple pipetting robots to workstations proteomics, automatically lead to sophisticated all the steps from the gel to the mass spectrometer. These systems usually consist of a spot picker, a digesting robot and a spotter.

The spot picker is programmed with a pick list that is generated in a 2D gel analysis program. According to this list, the device cuts the desired protein spots from a 2D gel and transfers them to a microtiter plate . There the digestion robot carries out the necessary steps for the in-gel digestion. The spotter then uses the peptide solution to generate the spots on the MALDI target or loads a suitable microtiter plate for automatic ESI-MS measurement. Manufacturers of automated 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 automation lies in the reduced need for personnel and the improved standardization of the process. With manual in-gel digestion, the results may depend on the experience of the user due to the many steps to be performed and there is a high risk of contamination. The improved quality of the results is therefore named as a main advantage of the fully automated processing of the samples.

Disadvantages of the automated solutions are the costs for robots, maintenance and the often proprietary consumables as well as the complicated operation of the systems. The automatic cutting out is dependent on digitized information of the spot position, which makes the use of gel analysis programs and special scanners necessary. This lengthy procedure and the (economic) need to run the system only with a certain number of samples (the smallest microtiter plate commonly used for this purpose holds 96 samples) prevents the spontaneous selection and analysis of a small number of spots from a single gel. Furthermore, the amount of data generated from the subsequent automated MS analysis must be viewed very critically, as its quality is often questionable and therefore requires careful evaluation, which takes considerably longer than collecting this data.

Low throughput

The disadvantages mentioned limit the reasonable use of automated in-gel digestion systems in routine laboratories, while research laboratories often stick to manual methods due to their need for a more flexible use of the instruments for protein identification . For this user group, the industry has developed kit systems for in-gel digestion.

Most of these kit systems are mere collections of chemicals and enzymes needed for in-gel digestion, and the method itself has not been changed. The advantage for the inexperienced customer is that the chemicals and enzymes as well as the protocol are obtained from a manufacturer, which guarantees the functioning of the method. Manufacturers of these kit systems are 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 in-gel digestion in order to achieve a simpler and standardized workflow without a robot. The Montage TM In-Gel Digest Kit from Millipore is based on the standard protocol for in-gel digestion, but relocates the process steps to a special microtiter plate, which means that a larger number of samples can be processed simultaneously. The solutions for the various steps are added by pipette, while the suction down through the bottom of the plate is carried out using a vacuum. This reduces the number of pipetting steps for the user because, on the one hand, there is no manual removal of the liquids and, on the other hand, they can be added using multi-channel pipettes or even pipetting robots. Some manufacturers have even adapted this system for use with their automated high-throughput systems. These details of the process make clear that the Millipore system is geared towards laboratories that have at least an average sample throughput.

The German company OMX GmbH has taken a completely different approach. The OMX-S ® product line is designed for the simultaneous processing of up to 24 samples, using a specially modified protocol as well as newly developed reaction vessels. The system arose from a critical analysis of the conventional protocol for in-gel digestion, where around 30 steps and 16 hours are estimated for the entire process from gel to peptide solution. The result of the investigation was a protocol shortened to four steps and about two hours. Process steps that do not lead to a significant improvement in the final peptide yield were omitted and the incubation time required for digestion was reduced by increasing the temperature. The special reaction vessels used in the system enable all steps of the in-gel digestion, from cutting to peptide extraction, to be carried out in one vessel. In this process, the gel spot is punched out with the integrated piercing tool and centrifuged into the reaction chamber, whereby it has to pass through a small opening and is thereby torn into small pieces. The gel remains in this reaction space for the entire process, only the solutions are added through the piercing channel and removed again by centrifugation. This procedure significantly simplifies and speeds up manual in-gel digestion, but since each sample has to be processed individually, processing larger batches is still very labor-intensive and in this respect cannot be compared with automated systems.

Individual evidence

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