Quantitative proteomics antibodies

PTMs of proteins enable many important functions in organisms (139). Proteins with PTMs can be enriched in a variety of ways antibody-based, chemical derivatization, and ionic interaction-based (89). Antibodies that are highly specific to a particular PTM site help functional assays greatly. Antibodies that preferably bind a class of PTM (e.g., proteins carrying phosphotyrosine residues) make possible the in-depth quantitative proteomic analysis of these proteins. The substoichiometric nature of PTMs yields a low concentration of modified proteins. Enrichment of these proteins allows for improved analysis. 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

Anderson NL, Anderson NG, Haines LR, et al. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA)./. Proteome Res. (2004) 3 235-244. 

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). 

Proteomics In MIcrotIuldIc Devices, Rgure 4 (a) Use of hydrodynamic flow fo create a microfluidic stream and direct it to specific areas of a larger channel, (b) The flow system is used to deposit biotinylated fluorescein in a grid pattern. The direction of flow during deposition is indicated by arrows, (c) Quantitation of C-reactive protein (CRP) in whole blood. Samples of blood were spiked with CRP and the microstream directed across a surface coated with anti-CRP before exposure with a Cy3-labeled secondary antibody. Reprinted from [24] under open access agreement with BioMed Central. 

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