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The in-gel digestion step primarily comprises the four steps; destaining, redox and alkylation (R&A) of the in the protein, proteolytic cleavage of the protein and extraction of the generated .
The destaining solution for CBB contains usually the buffer solution salt ammonium bicarbonate (NH4HCO3) and a fraction of 30%-50% organic compound solvent (mostly acetonitrile). 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 electrostatics bonds between the dye and the positively electric charge of the protein. In contrast to a mixture of water with organic solvent the effectivity of destaining is increased. An increase of temperature 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 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 of the silver attached to the protein by potassium ferricyanide or hydrogen peroxide (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 are complexed subsequently by sodium thiosulfate.
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 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.
An undesirable side effect of the use of proteolytic enzymes is the self digestion of the protease. To avoid this, in the past Calcium2+-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 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 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 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 of the gel pieces by treatment with acetonitrile 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 (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 proteins such as . Cleavable detergents are detergents that are cleaved after digestion, often under acidic conditions. This makes the addition of detergents compatible with mass spectrometry.
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, adsorption to the surface of and pipette tips, incomplete extraction of peptides from the gel and/or bad ionisation of single peptides in the mass spectrometer.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.
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.
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. This system simplifies the handling of the multiple pipetting steps by the use of multichannel 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.
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