DEPARTAMENTO DE DESARROLLO RURAL, MEDIO AMBIENTE Y ADMINISTRACION LO- LO-CAL

Prior to protein extraction, the mechanical disruption of cells (cell lysis) is required. Manual homogenization, vortexing, grinding, or liquid nitrogen treatment are the most used methods. Cell lysis depends on the presence or absence of cell walls. Especially difficult are plant cells where walls are made up with multiple layers of cellulose, particularly strong and difficult to disrupt [128]. Proper cell lysis is significantly important when intracellular proteins are studied. They can represent a tiny fraction of total cellular proteins and, thus, they are much more difficult to extract and recover [126, 129]. Proteins in biological samples are generally in a native state associated to other proteins and often being part of large complexes or membranes. Chemical and physical techniques can be applied to disturb cell walls. They can be grouped into five major categories: mechanical homogenization, osmotic and chemical lysis, ultrasounds or pressure, and temperature treatments. The application of two or more procedures has also been reported [130]. These methods must lyse rapidly and efficiently cells to extract proteins with minimal proteolysis or oxidation [126, 128].

Mechanical homogenization implies the use of a rotor-stator homogenizer or open blade mill. In the osmotic shock strategy, cells are suspended in a gently shaken hypertonic solution. Regarding chemical lysis, most common treatment includes the use of antibiotics, chelating agents, detergents, and solvents capable of disintegrating cells. For example, organic solvents (e.g. acetonitrile (ACN), ethanol (EtOH), and methanol (MeOH)) are efficient for destabilizing

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membrane bilayers [121]. Procedures employing detergents or strong chaotropic reagents (a substance that denatures and disrupts the structure of macromolecules, e.g. urea, thiourea or guanidine chloride) within the extraction buffer assure the disruption of interactions among proteins and between proteins and other compounds. Furthermore, low cost and easy to use detergents can efficiently disrupt cell membranes, break lipid-protein interactions, and solubilize proteins. Four different groups of detergents are mainly used for this puropose: bile acid salts, non-ionic, zwitterionic, and ionic. Bile acid salts (e.g. sodium deoxycholate) are charged soft detergents compatible with native protein extraction. Non-ionic detergents (e.g. Triton X series and Tween 20) are considered as mild since they disrupt protein-lipid interactions rather than inter/intra-protein interactions. Zwitterionic detergents (e.g. 3-[3- cholamidopropyl)dimethylammonio]-1-propanesulfonate) show intermediate properties and they can solubilize proteins more efficiently than non-ionic detergents. Ionic detergents (e.g. SDS) provide the harshest conditions and cause protein denaturation. SDS is considered the best protein solubilizer but it is incompatible with MS [131]. In fact, most traditional detergents and chaotropic reagents are not compatible with common MS ionization techniques and may interfer further enzymatic digestions. Therefore, their removal must be considered [121, 128]. Basic criteria for the selection of a suitable lysis buffer for protein extraction are buffer composition, pH, ionic strength, salt concentration, temperature, and presence of detergents, chaotropes, protein reducing agents (e.g. B-ME, dithiothreitol (DTT)), and presence of components preventing their proteolysis (e.g. protease inhibitors) [128, 129]. In conclusion, the selection of a sutaible lysis buffer or method is essential to avoid possible difficulties in next steps.

High intensity focused ultrasounds (HIFU) or pressure treatment involves the application of ultrasonic waves to the solution. Ultrasounds generate a cyclic sound pressure with a frequency greater than 20 kHz [132]. This pressure can accelerate certain chemical reactions, replacing traditional techniques or accelerating them. This phenomenon is produced by the focalization of high intensity ultrasonic waves that cross the liquid media and create an effect known as cavitation. Cavitation can be defined as a physical process by which numerous tiny gas bubbles are produced. Bubbles grow, oscillate, split, and implose [132, 133]. Therefore, these bubbles can be considered as microreactors inside which there are high temperatures and pressures. Ultrasonic devices (bath or probes) are commonly applied in many analytical laboratories. The greatest difference between them is that ultrasonic probe is inserted into the solution which provides, at least, 100 times greater energy than the ultrasonic bath [133, 134]. Sonication and

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high-pressure procedures have been applied to disrupt (lysis) different kinds of cells (microorganisms, plants, animal) and to extract organic and inorganic analytes from solid or liquid media [126]. Apart from protein extraction, the ultrasonic probe has also shown to reduce time in protein enzymatic digestion procedures [128].

After proteins extraction, the precipitation of proteins is very usual to separate them from interfering compounds, to change the surrounding environment if required in further steps, or to enrich proteins of interest. The precipitation of proteins can also be important when just the peptidome of a sample is studied, since it enables the removal of proteins [135]. It is a especially important step in the case of plant cell extracts, since they can contain high amounts of interfering molecules (polysaccharides, lipids, polyphenols, secondary metabolites, etc.). Removal of interfering compounds can be performed before or after the extraction procedure. Among various procedures, precipitation at pI, precipitation at high temperatures or precipitation using various reactants/solvents are the usual [135]. Lower protein solubility at pI can be explained by proteins zero net charge, which enables the association among protein molecules with a minimum charge repulsion. Thermal precipitation is based on the fact that proteins denature at high temperatures [135]. This method is also employed to stop enzymatic reactions. Moreover, various organic solvents such as acetone, EtOH, ACN, and their mixtures often provide effective protein removal. Additionally, protein deproteinization can be carried out by the addition of inorganic acids and salt solutions such as ammonium sulfate (salting out), trifluoroacetic acid (TFA), trichloroacetic acids, perchloric acid, or sulfosalicylic and alginic acids. In the analysis of food samples, precipitation under acidic conditions is more effective than with inorganic salts [136].