Proteome capturing

Antibody coverage of the human proteome is estimated to be about 5 to 10% of all human proteins and isoforms (Valle and Jendoubi, 2003). A major bottleneck in the use of protein expression arrays is the lack of such a comprehensive set of these capture agents (Hanash, 2003). Since an equivalent of the polymerase chain reaction (PCR) process for mass amplification of low abxmdant proteins does not exist, the remaining library of proteome capture ligands will need to be generated by other means such as recombinant protein expression systems (Cahill, 2001). 

FIGURE 9.1 Liquid chromatography workflow strategy options in proteomics. (a) bottom-up approach (b) top-down approach (c) selective sample cleanup directly combined with chromatographic separation (d) peptide capture with affinity restricted access material. 

Ornstein DK et al. Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines. Electrophoresis 2000 21 2235-2242. 

Patel V, Hood BL, Mohnolo AA, et al. Proteomic analysis of laser-captured paraffin-embedded tissues a molecular portrait of head and neck cancer progression. Clin. Cancer Res. 2008 14 1002-1014. 

Figure 19.2 A generalized proteomics work flow for the extraction and identification of proteins in FFPE tissue. Formalin-fixed tissues acquired by sectioning, needle dissection, or laser capture are deparaffinized in xylenes and are rehydrated in graded alcohols. The material is resuspended in buffer which generally contains a detergent/ protein denaturant and the sample is heated to complete the extraction process. The protein extract is reduced, alkylated, and digested with trypsin before protein profiling.

The preparation of particles or surfaces that are able to capture specifically a fraction of he proteome using metal affinity separations makes possible analysis of distinct protein... 

The sample materials from which proteins for proteomics studies may be extracted include fresh or snap-frozen cells from varied sources such as biological fluids, (serum, urine, plasma) and solid tissues such as biopsy specimens. Moreover, proteins isolated from ethanol-fixed paraffin-embedded tissues can be utilized for MS analysis.2 Protocols for the identification of proteins from formalin-fixed paraffin-embedded (FFPE) tissues have been recently developed.3 4 FFPE materials are the most common forms of biopsy archives utilized worldwide, and represent an important advancement for the large-scale interrogation of proteins in archival patient-derived materials. Finally, laser capture microdissected tissues have been successfully used for MS analysis.45... 

An alternative approach to assessing tissue-specific expression at the proteomic level can be achieved by MS of laser capture microdissected tissues.4 An important development in this arena is the ability to perform LCM and MS/MS on formalin-fixed paraffin-embedded tissues. 

However, recombinant antibodies may be less stable and have lower binding affinities than monoclonal antibodies (Valle and Jendoubi, 2003). Therefore, in order to fully implement the microarray format, a host of diverse capture agents could be required in addition to antibodies. These include peptides, small molecules, aptamers, ribozymes, or other molecular recognition probes yet to be discovered. However, it is also xmderstandable because of the diverse nature of proteins that additional technologies besides microarrays will be used in proteomics research (Hanash, 2003). 

The proteome may approach a million proteins. How will we be able to apply protein microarrays Where and how will we obtain the necessary libraries of capture ligands on such a scale Thomas Kodadek (2001) wrote. 

Siuti, N., Roth, M.f., Mizzen, C.A., Kelleher, N.L. and Pesavento, J.J. (2006) Gene-specific characterization of human histone H2B by electron capture dissociation. Journal of Proteome Research, 5, 233-239. 

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. 

Paweletz CP, Liotta LA, Petricorn EE (2001) New technologies for biomarker analysis of prostate cancer progression laser capture microdissection and tissue proteomics. Urology 57 160-163... 

VanMeter AJ, Rodriguez AS, Bowman ED et al (2008) Laser capture microdissection and protein microarray analysis of human non-smaU cell lung cancer differential epidermal growth factor receptor (EGPR) phosphorylation events associated with mutated EGFR compared with wild type. Mol Cell Proteomics 7 1902-1924... 

This is a reflection of the increasing knowledge of the complexity of in viva systems, and also the current powerful computational resources to capture, analyze, interpret and model those systems. In view of this complexity, not only the development of genomics, proteomics and metabonomics databases, but also the development of systems biology methods helps us to understand the underlying mechanisms in any given ADME process [80]. 

Banks BE, Dunn MJ, Eorbes MA, et al. (1999) The potential use of laser capture microdissection to selectively obtain distinct populations of ceUs for proteomic analysis - preliminary findings. Electrophoresis 20, 689-700. 

Li C, Hong Y, Tan YX et al. Accurate qualitative and quantitative proteomic analysis of clinical hepatocellular carcinoma using laser capture microdissection coupled with isotope-coded affinity tag and two-dimensional liquid chromatography mass spectrometry. Mo/ Cell Proteomics 2004,3399-409. 

Examine the utility of newer technologies related to gene expression (e.g., gene arrays, proteomics, laser capture microscopy) to aid in the understanding and elucidation of the underlying... 

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