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Digestion (in-gel or in-solution)

Among the endoproteases which could be used for protein digestion, the serine protease trypsin is most commonly employed as it generates peptides which are highly amenable to MS(/MS) analysis. Depending on the preceding workflow, the enzymatic digestion of proteins is performed either in-gel or in-solution, generally, the in-gel digestion methodology has become routine for proteins separated by 2-D electrophoresis while in-solution digestion are usually used in LC-MS/MS analysis. The protein is cut enzymatically into a limited number of shorter fragments during digestion and these fragments are called peptides and allow for the identification of the protein with their characteristic mass and pattern.

In-Gel Digestion

Following the separation of samples by 1-D or 2-D gel electrophoresis, proteins are fixed and visualized using an MS-compatible stain, usually Coomassie Blue, or silver employing a glutaraldehyde-free protocol. Visualized proteins are excised from the gel and the respective gel bands or spots are washed for destaining and dehydrated before being trypsinized.

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. Different studies about the penetration of the enzymes to the gel showed the process to be almost completely driven by diffusion, by cutting the gel to pieces as small as possible the efficiency of the in-gel digestion could be achieved.

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 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.

In order to ensure efficient hydrolysis of those proteins embedded in the gel matrix, a relatively high enzyme concentration is generally used. The generated proteolytic peptides can subsequently be released from the gel matrix, by for example 50% acetonitrile (ACN)/5% formic acid (FA) being used as an extraction buffer, in combination with sonication. To meet the requirements of peptides with different physical and chemical properties an iterative extraction with basic or acidic solutions is performed.

The efficiency of in-gel digestion and peptide extraction depends on a variety of factors, including: (i) the physico-chemical properties of the proteins and resulting peptides (e. g. , degree of hydrophobicity, size, amino acid sequence); (ii) the composition, size, and thickness of the gel pieces; (iii) the composition of the extraction buffer (e. g. , acetonitrile (ACN) concentration); (iv) the type of enzyme and its specific activity; (v) the general reaction conditions (e. g. , temperature, time, ratio of enzyme to substrate); (vi) the type of protein stain (e. g. , Coomassie or silver).

One significant advantage of in-gel protein digestion is that any contaminants (e. g. , detergents, salts) are already removed during electrophoresis, and that the generated peptide samples can be readily subjected to (LC/) ESI-MS analysis. However, the effectiveness of this procedure can be limited due to poor accessibility of the protease to proteins and/or inefficient release of peptides from the gel matrix, as well as inadequate storage of gels. As an excellent alternative to gel electrophoresis combined with in-gel digestion, proteins can be directly digested in-solution, which is usually followed by 1-D or 2-D LC to effectively separate the resulting peptide mixture before ESI-MS.

In-Solution Digestion

For in-solution digestion, a compromise between protein solubilization and enzyme activity must be reached. In the reduction and alkylation (r&a) of the cystines or cysteines potentially embodied in the protein, the disulfide bonds of the proteins are irreversibly broken up and the optimal unfolding of the tertiary structure is obtained. 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. With denaturing electrophoresis it is strongly recommended to perform the reaction before the execution of the electrophoresis, since there are free acrylamide monomers in the gel able to modify cysteines.

Detergents should be generally avoided but, depending on the physico-chemical characteristics of the proteinlysate, organic solvents and/or chaotropes may be used. For the analysis of proteins which are difficult to solubilize or denature (e. g. , membrane proteins), 8M urea may be used in conjunction with the resilient protease Lys-C; subsequently, trypsinization is performed at a reduced concentration of chaotrope (2M urea). Following acidification using FA it may be possible directly to perform LC/ESI-MS, although it is generally recommended that an additional desalting step be performed. As a promising alternative to double proteolytic digestionin urea, organic solvents can be used to solubilize and effectively digest hydrophobic proteins in-solution. In order to enhance proteolytic digestion, enzymes that are chemically immobilized or physically adsorbed to a stationary phase can be used.

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