Proteomics methodology

X. Peng, X. Ye, and S. Wang. Identification of Novel Immunogenic Proteins of Shigella Flexneri 2a by Proteomic Methodologies. Vaccine, 22(2004) 2750-2756. 

Zhang HT, Kacharmina JE, Miyashiro K, Greene MI, Eberwine J. Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution. Proc Natl Acad Sri USA 2001 98(10) 5497-5502. 

Recent advances in proteomics methodologies make them a promising strategy for food science studies and are used by research institutions, industries, agencies, and regulatory laboratories, combining efforts to acquire the needed knowledge on food composition, quality, and safety. Very useful reviews on proteomics applications in food science have been recently published (9,10). 

In view of this, our group has greatly contributed in the application of proteomics methodologies for the assessment of the quality and safety of seafood products (11-14). As an example, Figure 1 summarizes the proteomics pipeline and tools that are currently being used at our laboratory for fish authentication (12,14,15). Two consecutive phases, discovery phase and target-driven phase are described in Sections 2.1 and 2.2. 

Advances in genomics and proteomics methodologies, directed at identifying those genes expressed at elevated levels in tumors compared to normal tissues, have led and accelerated the identification process of many potentially useful tumor biomarkers for the diagnosis and therapy. 

Charlwood, J., Flanrahan, S., Tyldesley, R., Langridge, J., Dwek, M., Camilleri, P. 2002. Use of proteomic methodology for the characterization of human milk fat globular membrane proteins. Anal. Biochem. 301, 314-324. 

Eikmans, M., Baelde, H. J., de Heer, E., and Bruijn, J. A. RNA expression profiling as prognostic tool in renal patients toward nephrogenomics. Kidney Int. 62,1125-1135, 2002. Gibbs, A. Comparison of the specificity and sensitivity of traditional methods for assessment of nephrotoxicity in the rat with metabonomic and proteomic methodologies. J. Appl. Toxicol. 25, 277-295, 2005. 

Authors come from a broad variety of affiliations, be it big industries, small and half-way grown up start-ups, clinics and academia. All of them integrate and focus on the question how proteomics methodology can be improved and serves best in their field of expertise. 

Although MS is most often used for qualitative analysis and structural determination, several methods have been developed for the quantitative assessment of proteins, through peptides or glycopeptides, in order to determine the extent of their over- or under-expression in cellular systems. Some proteomic methodologies use natural isotope incorporation to prepare internal standards for isotope dilution experiments. The protein concentration may then be determined by quantifying the abundances of either the peptides or glycopeptides. These methods were summarized and compared in a review produced in 2007 [119]. 

The availability of genome information for a particular pathogen allows the adaptation of bottom-up [36,37] or top-down [38 0] proteomics methodologies for microorganism identification by MS [41 5] (Fig. 3). These proteomics-based approaches are based on the initial identification of one or more individual protein biomarkers (from, e.g., their corresponding tryptic peptides and/or tandem mass spectra). By inference, the microorganism from which these proteins originate is then identified. For instance, rapid in situ (on a MALDI sample slide) proteolysis of... 

FIGURE 46.2 A major technique used in proteomics methodology for PTSD diagnosis. (1) Two-dimensional gel electrophoresis for proteomics 2D-D1GE proteomes mixed in gel (2) shotgun proteomics methodology. 

Von Haller, P.D., Yi, E., Donohoe, S., Vaughn, K., Keller, A., Nesvizhskii, A.I., Eng, J., Li, X.J., Goodlett, D.R., Aebersold, R., Watts, J.D. (2003). The Application of New Software Tools to Quantitative Protein Profiling Via Isotope-coded Affinity Tag (ICAT) and Tandem Mass Spectrometry II. Evaluation of Tandem Mass Spectrometry Methodologies for Large-Scale Protein Analysis, and the Application of Statistical Tools for Data Analysis and Interpretation. Mol. Cell. Proteomics 2, 428 -42. 

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. 

For a proper understanding of the protein binder s function in artworks and historical building materials, it is essential to identify the individual proteinaceous additives and to distinguish them even in materials where they are present in very small amounts, in insoluble forms and often in matrices unsuitable from an analytical point of view. For many materials the appropriate analytical methodology has not been found up to now and, in general, this type of analyses has not become a routine technique. Thanks to the recent development of proteomics (Section 6.2) most of the afore-mentioned problems have been resolved mass spectrometry forms a fundamental platform for this new methodology. 

---