Protein analysis strategies

This chapter will address the applications of protein-based bioinformatics to analysis of microorganisms introduced intact into the instrumental system for rapid processing and analysis. Strategies that require offline extraction and fractionation of proteins will not be discussed. Although the amplification of nucleic acids is a powerful approach, especially coupled with mass spectrometry,15 it requires extraction and processing, and thus is not included. 

Fenselau, C. MALDI MS and strategies for protein analysis. Anal. Chem. 1997, 69, 661A-665A. 

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. 

Hoffman, K. (2000). Strategies for host cell protein analysis. Biopharm-Appl TBio 13(5) 38 15. 

C. Fenselau, MALDI MS and Strategies for Protein Analysis, Anal. Chem. 1997,69, 661 A R. W. Nelson, D. Nedelkov, and K. A. Tubbs, Biomolecular Interaction Analysis Mass Spectrometery, AnaL Chem. 2000, 72, 405A J. J. Thomas, R. Bakhtiar, and G. Siuzdak, Mass Spectrometry in Viral Proteomics, Acc. Chem. Res. 2000,33, 179 A. P. Snyder, Interpreting Protein Mass Spectra (Washington, DC American Chemical Society, 2000) S. C. Moyer and R. J. Cotter, Atmospheric Pressure MALDI, Anal. Chem. 2002, 74, 469A. 

Figure 4.2 Schematic overview of two protein identification strategies commonly followed in proteomics. Protein samples are separated by either two-dimensional (2-D) or one-dimensional (1 -D) polyacrylamide gel electrophoresis (PAGE). In both strategic tracks, proteins are converted into a set of peptides by enzymatic digestion (e.g., with trypsin) prior to MS analysis. Peptide mass fingerprinting (PMF) by MALDl MS is predomi-...

While all these strategies rely on specific properties of phosphopeptides in MS analysis, a more global approach involving shotgun protein identification strategies and SEQUEST database searching (Ch. 18.3.2) is demonstrated by the group of Yates for protein complexes and lens tissue proteins [29]. 

The equalization approach is based on the opposite strategy, in that only low-abundance proteins are collected and subjected to LC-MS analysis. The method relies on bead-bound combinatorial peptide ligand libraries (10) that enrich low-abundance proteins. This strategy is much cheaper than antibody precipitation, but it has the clear drawback that only proteins that have affinity to one of the millions of beads present in the library used can be equalized for further analysis. 

Proteins have been a major preoccupation of biochemistry and molecular biology since the inception of those scientific fields. There are several ways to study proteins. Analysis of three-dimensional structure has been briefly described above. Biochemists have devised many ways to study cn/ymc function. One classic technique is to isolate an enzyme to near purity, then to attempt to study the chemical reaction it mediates in great detail. This is a key strategy in dissecting the biochemical pathways of energy metabolism, biosynthesis, and degradation. Another technique is made possible by modem molecular genetics. 

Monoclonal gammopathy is related to the presence in the blood of a monoclonal Ig (Sahota et al, 1999). Serum protein analysis should be done when multiple myeloma, Waldenstrom s macroglobulinemia, primary amyloidosis or a related B cell disorder is suspected (Attaelmannan and Levinson, 2000). The strategies to detect as well as to identify monoclonal gammopathies in the clinical laboratory have been reviewed (Keren et al, 1999 Kyle, 1999b). Other, but not routinely used electrophoretic techniques, have been employed to further characterize monoclonal Igs, including isoelectric focusing and 2-DE. 

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