Enrichment techniques, proteomics

Zhao, Y. and Jensen, O.N. (2009) Modification-specific proteomics strategies for charactmzation of post-translational modifications using enrichment techniques. Proteomics 9,4632-4641. 

The proteomic analysis of the brain has certain limitations that are related either to the sample and/or analytical approach. In the analysis of the brain, many factors may be involved, such as differences among individuals, differences in age and sex, possible other diseases, treatment with medicines, as well as technical factors, disease-unrelated factors, such as postmortem time, improper treatment of the samples, etc., all of which can affect a clear discrimination between healthy and diseased states of interest. The technical limitations involve inefficient detection of low-abundance gene products, hydrophobic proteins (they do not enter the IPG strips), and acidic, basic, high-, and low-molecular mass proteins. All these protein classes are underrepresented in 2-D gels (Lubec et al., 2003 Fountoulakis, 2004). A combination of proteomics methods with protein separation, enriching techniques, and alternative methodologies for detection will improve the detection of additional differences between AD and control brains. Such differences may be essential in the discovery of early disease markers and therapeutic approaches. 

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). 

As outlined above displacement chromatography may find its most important uses in the analytical area. The ability to enrich trace levels of components is ideally suited to the proteomics where more powerful tools are desperately needed to address the vast concentration ranges present in order to identify trace components. The technique also offers a way to isolate large quantities of protein variants which is important for the identification and characterization of minor product-related impurities commonly associated with therapeutic proteins. 

The high sample demands and low-throughput of LC-MS methods have led to the creation of a capillary electrophoresis (CE) platform for ABPP [48]. Proteomes are labeled with a fluorescent probe, digested with trypsin, and enriched with antifluorophore antibody resins. Use of CE coupled with laser-induced fluorescence (LIF) detection to analyze the enriched peptides resulted in far superior resolution to ID SDS-PAGE, particularly for enzymes that share similar molecular masses. Sensitivity limits of 0.05-0.1 pmol/mg proteome, negligible sample requirements (—0.01—0.1 pg proteome), and the ability to perform rapid CE runs in parallel with 96-channel instruments, make CE-based ABPP a potentially powerful technique. One drawback is that the identities of the probe-labeled proteins are not immediately apparent, and correlated LC-MS experiments must be performed to assign protein identities to the peaks on the CE readout. 

D polyacrylamide gel electrophoresis (2D PAGE) and MS are weU-established and the most commonly employed techniques in proteomics today. 2D PAGE, however, provides limited information of the total amount of proteins. Low-abimdance proteins and small peptides are not detected [1]. Additional methodologies and techniques in sample preparation, selective enrichment, high resolution separation, and detection need to be developed which would allow even higher resolution than 2D PAGE. Acceptable sensitivity to detect the low-abundance proteins is also still an issue. LC can address some of the above-mentioned... 

Sample complexity reduction strategies are required to enable neuroscientists to address the issues mentioned above, and facilitate meaningful applications of proteomic analyses. The feasibility of protein enrichment by subcellular fractionation has been demonstrated through the analysis of the rat brain sub-proteomes of cytosolic, mitochondrial and microsomal fractions (Krapfenbauer et al. 2003). Another well-established subcellular fractionation technique, synaptosomal isolation, has recently... 

Co-immunoprecipitation is a common technique to enrich protein interaction complexes. This method uses an antibody to bind a known protein, which in turn has known or unknown binding partners. Shotgun proteomics is then performed to determine the enriched proteins. Quantitative MS is used to validate bound proteins and measure binding stoichiometry for proteins in the complexes prepared via IP (127,128). Quantitation approaches used include SILAM (129), label-free (130), and AQUA peptides with a normalization step (131). 

These recent studies have concluded that there is an inherent limitation of two-dimensional gels, namely that of maximum load compared to the range of protein abundances, which precludes low abundance protein identification. It would appear from these conclusions that, to be useful in detecting any protein with less than moderate abundance, 2DE must be applied to subfractionated or otherwise enriched samples, hi fact, the same groups have already started using and advocating mass spectrometry-based techniques for proteome comparison, bypassing completely the 2DE step. ... 

Knowledge about the subcellular localization of a protein may provide a hint as to the function of the protein. The combination of classic biochemical fractionation techniques for the enrichment of particular subcellular structures with the large-scale identification of proteins by mass spectrometry and bioinformatics provides a powerful strategy that interfaces cell biology and proteomics, and thus is termed subcellular proteomics. ... 

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