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Chromatographic methods are varied and allow the separation of analytes in complex mixtures in function of their distribution between two phases: a stationary phase and a mobile phase that percolates through the stationary phase. The analytes enter the column with the mobile phase, and migrate at different rates, depending on their affinity for each of the phase,

Sucrose, sorbitol, mannitol, EDTA, proteases inhibitors Homogenization Centrifugation Rate zonal

centrifugation centrifugation Differential centrifugation Isopycnic Separation as thin bands

in a density gradient Separation of particles by size and density Mostly used for nucleic acids not affected by the size

which provides separation. Many types of chromatography have been used, and only the principle of some will be shortly described. For extended review, see reference69.

Ion-exchange chromatography uses stationary phases that bind proteins according to their charge. The elution is performed with increasing salt concentration buffers. The non- denaturing conditions limit the analysis to soluble proteins only. Fountoulakis et al.70 successfully detected low-abundant proteins of the bacterium H. Influenzae. Strong cation exchange belongs to this category of chromatography and has been widely-used as a first dimension separation for proteomics,71-73 or in MudPit as mentioned in section 3.

Reverse Phase liquid chromatography (RP-LC) separates proteins according to their hydrophobicity. Proteins are adsorbed on a stationary phase carrying hydrophobic groups, and are eluted with increasing concentration of acetonitrile. It is one of the most widely used type of chromatography in proteomics, namely in shotgun multidimensional strategies. Normal phase chromatography (polar stationary phase and mobile phase non-polar, in contrast to reversed-phase) is not so much used in proteomics, due to the poor compatibility of normal phase solvent and ESI-MS and low reproducibility compared to RP-HPLC. Recently however, it has become useful as chiral chromatography technique, to analyze enantiomeric bioactive lipids, using electron capture atmospheric chemical ionization/tandem mass spectrometry.74

Affinity chromatography is based on the interaction between a particular compound constituting the stationary phase, and a subset of proteins. The nature of the compound used determines the range of proteins that bind to the column. For example, monoclonal antibody will bind a single protein, heparin and hydroxyapatite phase will bind thousands of proteins.

Heparin affinity chromatography uses gels containing heparin, a natural mixture of linear polymeric sulfated glycosaminoglycan, which has the highest negative charge density observed in biological molecule. This property also makes it a strong cation exchanger (SCX),

with affinity for a broad range of proteins, such as coagulation factors, nucleic acid-binding proteins (protein synthesis factors) or growth factors. An illustration of this technique is the work of Fountoulakis et al.,75 who separated the soluble proteins of H. Influenzae and showed the enrichment of low-abundant proteins.

Figure 9: Heparin

Hydroxyapatite affinity chromatography uses a matrix carrying positively charged (calcium) and negatively charged (phosphate) sites. Proteins are retained in two ways, either by non-specific electrostatic interactions between their positive charges and the general negative charge of the hydroxyapatite when equilibrated in phosphate buffer, or by complexation of the proteins carboxyl sites with the calcium sites. Elution is performed by increasing salt concentration buffer. Fountoulakis et al. showed the fractionation of E. coli soluble proteins.76

More interestingly, immunoaffinity columns are increasingly used for the depletion of high abundance proteins, to enhance sensitivity in proteome analysis, especially when dealing with plasma or serum samples (high complexity). This type of column was shown to be particularly useful for the detection of biomarkers in plasma.77, 78 Currently, there are three multi-parameter depletion resins commercially available: (a) the multiple affinity removal system (MARS) from Agilent Technologies, targeting 6 abundant plasma proteins (b) an IgY- based immunoaffinity resin against 12 individual proteins developed by Genway and now

commercialized by Beckman Coulter for the ProteomeLab IgY system, and (c) the ProteoPrep 20 immunodepletion kit from Sigma, to remove 20 different plasma proteins.

Size-exclusion chromatography separates proteins according to molecular mass, like the second dimension of 2D-GE. However, the main difference is the non-denaturing conditions of the chromatography, allowing studying protein complexes.79 This technique is also called gel filtration and uses dextran derivatives-gels (Sephadex gels). A recent illustration of the technique is Hu.80

A summary of these approaches and the physicochemical properties underlying the separation process is given in Table 1.

Table 1: Summary of fractionation methods and the physicochemical properties according to which the

separation is performed.

Fractionation method Physicochemical properties

Ultracentrifugation Density SCX, Ion-Exchange Chromatography Charge

Reverse phase Chromatography (RP) Hydrophobicity Affinity Chromatography

(heparin, hydroxyapatite)

Specific biomolecular interactions (Affinity + Charge) Size Exclusion Chromatography MW (Stokes radius)

Isoelectric focusing pI

Gel electrophoresis MW (Stokes radius)

One can also distinguish between analytical and preparative chromatography. Preparative elution chromatography is generally carried out under mass overload: the sample concentration is increased beyond the linear adsorption region, resulting in asymmetric band profiles, while analytical chromatography remains in the linear adsorption range. The main

difference lies in the working flow rates (a few up to 30 mL/min in preparative mode, and a few µL/min in the analytical mode) and the collection of fractions (preparative) or not (analytical).

As a conclusion, chromatographic methods can be powerful tools for enrichment of low-abundance proteins prior to 2DE. However, the enrichment of abundant proteins is achieved simultaneously as for low-abundant ones. No clear correlation exists between the elution profile and a particular functional class of proteins. And the main drawback is the protein loss due to adsorption inherent to the technique. In addition, the sometimes large amount of salts (depending on which chromatography is used) and the large volumes of eluted fractions constitute significant challenges to the subsequent analysis of chromatographic fractions. Concentration and desalting steps are thus necessary, increasing the risk of protein loss.

In addition to prefractionation use, chromatographic methods have also been used in a two dimensional approach (MudPit, typically combination of strong cation exchange with RP). These powerful methods represent a way to overcome the limitations of 2DE, particularly for high MW and hydrophobic proteins. However, some limitations remain. Highly hydrophobic proteins are difficult to digest and necessitate additional cleavage steps. Low MW proteins are also a challenge due to the insufficient number of peptides available for MS analysis.