Proteomics protein solubilization

Luche, S., Santoni, V. and Rabi I loud, T. (2003) Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. Proteomics 3, 249-253. 

Another limitation of 2D gels is that membrane proteins are underrepresented. Because membrane proteins account for approximately 30% of total proteins (Wallin and Von Heijne, 1998), this is a serious problem for characterization of the proteome. The relative lack of membrane proteins resolvable on 2D gels can be attributed to thee main factors (i) they are not abundant, and therefore are difficult to detect by standard staining techniques, (ii) they often possess alkaline pi values, which make them difficult to resolve on the pH gradients most often used for isolelectric focusing, and (iii) the most important reason for under representation may be that membrane proteins are poorly soluble in the aqueous media used for isoelectric focusing (Santoni et al., 2000). Membrane proteins are designed to be soluble in lipid bilayers and are therefore difficult to solubilize in water-based solutions. 

Here again, not all chromatographic setups are usable for any proteomics question. The use of protein reverse phase chromatography, which has been advocated for plasma proteomics (Moritz et al. 2005), precludes in turn the use of any detergent of any type. This prevents the use of this chromatographic setup in most subcellular proteomics experiments, where detergents must be used to solubilize the membrane limiting the subcellular compartments. 

It should be obvious from the above that membrane proteomics strictly follows Murphy s law. This is due to the fact that there is a mutual exclusion, for physicochemical reasons, between on the one hand the conditions that must be used to solubilize in water all membrane proteins, including the most hydrophobic ones, and thus give a fair representation of the protein population in the sample, and on the other hand the conditions prevailing in the high resolution peptide separation methods. On top of this problem, there is a second problem linked to the membrane versus aqueous phase volume in many subcellular preparations, which makes transmembrane proteins rare compared to water-soluble proteins. 

Li, N., Shaw, A.R., Zhang, N.,Mak,A. andLi, L.(2004)Lipidraftproteomics analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry. Proteomics 4, 3156-3166. 

The first step in the analysis of proteomes and phosphopro-teomes involves extraction of proteins from the biological system under study the objective is to solubilize the proteins and to prepare them for subsequent analysis. Obviously, this step is critical for the overall success of the analysis, and choice of... 

Generically, sample preparation in bottom-up proteomics involves the solubilization of proteins from the biological source, protein fractionation, subsequent enzymatic digestion of the proteins, and separation of the resulting peptides. Unique enrichment techniques can be applied in many of these preparatory steps (Figure 5). 

Figure 9.23 Gel-based Membrane Proteomics. Membrane proteins can be analysed by a four-step process of solubilization, separation, digestion/extraction, and finally MS identification (illustration from Wu and Yates, 2003, Fig. 1).

The characterization of complex proteomes requires separation, detection and analysis of many thousands of proteins from whole cells, tissues or organisms. Cellular/tissue proteins are solubilized (Herbert, 1999) prior to their separalion/analysis. Two-dimensional gel electrophoresis has dononstrated the potential to separate several thousand of proteins (Klose 1999 RabiUoud, 2(XK)) in single experiment and has been the method of choice for the separation/purificalion in proteomic studies. 

Accurate modeling of microbial solubilization of lignocellulose will be dependent on knowledge of the dynamics of microbial cell concentration over the course of bioconversion. While measurement of cell concentrations distinct from the concentration of substrate is trivial for soluble substrates, it is a substantial and not-yet-resolved challenge for fermentation of particulate substrates based on plant cell walls. Cell measurement has been approached on the basis of elemental composition (pellet nitrogen, [25]), concentration of cellular macromolecules (total protein [26] or DNA via quantitative PCR [27]), and estimated by indirect methods, such as off-gas analysis [25] and detection of enzymes (ELISA assays [28]). Future efforts using quantitative proteomics approaches also hold promise. 

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