Proteomic analysis proteins

FIGURE 15.1 Overview of configuration of MS and MS-based proteomic analysis. Proteins are extracted from biologic samples and fractionated by a variety of separation methods including gel separation, HPLC, and capillary electrophoresis. The common ion sources and mass analyzers used are indicated. 

Griffin, T. J. Han, D. K. Gygi, S. R Rist, B. Lee, H. Aebersold, R. Parker, K. C. Toward a high-throughput approach to quantitative proteomic analysis Expression-dependent protein identification by mass spectrometry. J. Am. Soc. Mass. Spectrom. 2001,12,1238-1246. 

Hoffmann, R, Ji, H., Moritz, R. L., Connolly, L. M., Frecklington, D. F., Layton, M. J., Eddes, J. S., Simpson, R. J. (2001). Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215 a non two-dimensional gel electrophoresis-based proteome analysis strategy. Proteomics 1(7), 807. 

The enormous dynamic range of proteins in the sample represents an additional difficulty in proteome analysis. The best example is semm with a protein abundance ranging over eleven orders of magnitude (Anderson and Anderson, 2002). To detect the low abundant species, one has to load a sufficient amount of digest on a column to meet the limit of detection (LOD) of the MS instrument. Some reports published used up to 2.5 L of plasma with an extensive fractionation of intact proteins prior to LC-MS analysis on the peptide level (Rose et al., 2004). 

The combination of this top-down proteomics approach, which generates information on the structure of the intact protein, with a bottom-up approach for protein identification (using MS/MS data of tryptic peptides from the collected fractions) has been particularly useful for identifying posttranslational modifications, cotransla-tional processing, and proteolytic modifications in a number of proteins. Examples from our work will be shown to illustrate this hybrid methodology for proteomics analysis. 

Wang, H., Hanash, S. (2005). Intact-protein based sample preparation strategies for proteome analysis in combination with mass spectrometry. Mass Spectrom. Rev. 24, 413 126. 

Pennington, S.R., Wilkins, M.R., Hochstrasser, D.F. and Dunn, M.J. (1999) Proteome analysis from protein characterization to biological function. Trends in Cell Biology, 1, 168-173. 

Simpson RJ et al. Proteomic analysis of the human colon carcinoma cell line (LIM 1215] development of a membrane protein database. Electrophoresis 2000 21 1707-1732. 

Edgar PF Comparative proteome analysis. Tissue homogenate from normal human hippocampus subjected to two-dimensional gel electrophoresis and Coo-massie blue protein staining. Mol Psychiatry 2000 5 85-90. 

Arnott D et al. An integrated approach to proteome analysis identification of proteins associated with cardiac hypertrophy. Anal Biochem 1998 258 1-18. 

Ping P et al. Functional proteomic analysis of protein kinase C epsilon signaling complexes in the normal heart and during cardioprotection. Circ Res 2001 88 59-62. 

Reinheckel T et al. Adaptation of protein carbonyl detection to the requirements of proteome analysis demonstrated for hypoxia/reoxygenation in isolated rat liver mitochondria. Arch Biochem Biophys 2000 376 59-65. 

Weekes J et al. Bovine dilated cardiomyopathy proteomic analysis of an animal model of human dilated cardiomyopathy Electrophoresis 1999 20 898-906. Doherty NS et al. Analysis of changes in acute phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two-dimensional gel electrophoresis. Electrophoresis 1998 19 355-363. 

Journet A et al. Towards a human repertoire of monocytic lysosomal proteins. Electrophoresis 2000 21 3411-3419. Soskic V et al. Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor. Biochemistry 1999 38 1757-1764. Thiede B et al. A two dimensional electrophoresis database of a human Jurkat T-cell line. Electrophoresis 2000 21 2713-2720. 

Hirose I et al. Proteome analysis of Bacillus subtilis extracellular proteins a two-dimensional protein electrophoretic study. Microbiology 2000 146 65-75. 

Malhotra S et al. Proteome analysis of the effect of mucoid conversion on global protein expression in Pseudomonas aeruginosa strain PAOl shows induction of the disulfide bond isomerase, dsbA. J Bacterid 2000 182 6999-7006. 

Kaufmann H et al. Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. Biotechnol Bioeng 1999 63 573-682. Kristensen DB et al. Analysis of the rat dermal papilla cell proteome. Exp Dermatol 1999 8 339-340. 

Chevalier S et al. Proteomic analysis of differential protein expression in primary hepatocytes induced by EGF, tumour necrosis factor alpha or the peroxisome pro-liferator nafenopin. Eur J Biochem 2000 267 4624-4634. 

Witzmann FA et al. Toxicity of chemical mixtures proteomic analysis of persisting liver and kidney protein alterations induced by repeated exposure of rats to... 

Formaldehyde fixes proteins in tissue by reacting with basic amino acids— such as lysine,5 7—to form methylol adducts. These adducts can form crosslinks through Schiff base formation. Both intra- and intermolecular cross-links are formed,8 which may destroy enzymatic activity and often immunoreactiv-ity. These formaldehyde-induced modifications reduce protein extraction efficiency and may also lead to the misidentification of proteins during proteomic analysis. 

Jiang X, Jiang X, Feng S, et al. Development of efficient protein extraction methods for shotgun proteome analysis of formalin-fixed tissues. J. Proteome Res. 2007 6 1038-1047. 

PROTEOMIC ANALYSIS OF PROTEIN EXTRACTED FROM TISSUE/CELLS... 

A study of by Palmer-Toy et al.,12 summarized in Table 19.1, provides further empirical evidence of the utility of techniques coupling heating with efficient protein extraction for the proteomic analysis of FFPE tissue. A specimen from a patient with chronic stenosing external otitis was divided in half and preserved as fresh-frozen tissue or FFPE. Ten micromolar sections of the FFPE tissue were vortexed in heptane to deparaffinize the tissue and were then co-extracted with methanol. The methanol layer was evaporated, and the protein residue was resuspended in 2% SDS/lOOmM ammonium bicarbon-ate/20mM dithiothreitol (DTT), pH 8.5 and heated at 70°C for lh. After tryptic digestion, 123 total confident proteins were identified in the FFPE tissue, compared to 94 proteins identified from the fresh-frozen tissue. Hwang et al. also reported up to a fivefold increase in protein extraction efficiency for samples extracted in a Tris-HCl/2% SDS/1% Triton X-100/1% deoxycholate solution at 94°C for 30 min versus samples extracted in 100 mM ammonium bicarbonate/30% acetonitrile at the same temperature.14... 

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