Proteins expression profiling

Global proteomic profiling by MS is gaining significant attention as a tool for discovering disease biomarkers. Two basic approaches have been explored. With the first, MS analysis is performed with a material from a specific disease condition and the mass spectra are compared to those of normal individuals or related disease conditions. SELDI-TOF MS gained popularity in this area because of its simplicity and the requirement for only small amounts of samples.38 13 In MS-pattem based disease categorization, the mass spectral patterns are considered reflective of the proteins present in samples from distinct clinical conditions. 

Mixtures of principal components in the presence of several minor components are common with spot sampling from 2-DGE gels. Since 

MALDI-TOF provides limited capabilities for mixture analysis, LC/MS methods are used to provide more detailed interrogation of protein expression and peptide sequence. The use of LC/MS approaches for protein identification in conjunction with 2-DGE offers distinct advantages such as the ability to handle low picomole (miniaturized) level samples, enhanced separation, detection, the amenability to N-terminally blocked proteins, and fast analysis. The LC/MS methods for protein characterization focus on four distinct goals (1) confirmation of putative sequence, (2) identification of amino acid modifications, (3) identification of known proteins, and (4) sequence determination of unknown proteins. 

The development and use of various protein sequence databases for automated search routines (Eng et al., 1994 Clauser et al., 1999) are an essential component of protein analysis that uses mass spectrometry techniques. These programs (i.e., SEQUEST, MASCOT) require only a few peptides for matching therefore, the absence of a match for a particular peptide does not affect the search performance. Using protein database searches provides an efficient way of confirming a putative sequence from corresponding full-scan mass spectrometry and MS/MS data. 

The capillary LC/MS-based approach for peptide mapping performed by Arnott and colleagues features miniaturized sampleloading procedures, which are routinely amenable to small quantities of peptides. The reliable characterization of protein/peptide mixtures in conjunction with the widely used 2-DGE methods offers a powerful fingerprinting approach in the pharmaceutical industry. Low femtomole detection limits (typically 50 femtomole) with a mass accuracy of +0.5Da provide unique advantages for protein identification. Liberal parameters for mass range and unmatched masses are used for the initial protein search, whereas more conservative parameters are used to reduce the number of matches and to improve the confidence in the search. 

The use of high performance liquid chromatography (HPLC) on-line or off-line is an essential feature for peptide mapping to integrate the removal of buffers and salts (purification) and the separation of analytes (preconcentration) with mass spectrometry. With on-line LC/MS approaches, low flow rates ( 100pL/min) have been demonstrated to provide maximum sensitivity with ESI techniques for the analysis of proteins. In the work performed by Arnott and 

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Imaging mass spectrometry involves MS performed on tissue sections mounted on a MALDI plate. The mass spectra generate images and an in situ protein expression profile of the specimen is analyzed. Specifically, the frozen tissue sections applied to a MALDI plate are subjected to laser interrogation and analyzed at regular spatial intervals. The mass spectral data obtained at different intervals are compared to generate a spatial distribution of masses (proteins) across the tissue section. 

FIGURE 15.3 Outline of experimental protocol used for ICAT differential protein expression profiling. Protein mixtures from two cell populations are labeled with light or heavy isotopic versions of a cleavable ICAT reagent. Labeled proteins are combined, subject to multidimensional separation by SCX, RP, and avidin affinity chromatography, then analyzed by tandem MS for peptide and protein identification. Based on the relative ratio of the two isotopically labeled peptides, a relative abundance of protein expression can be determined. 

Typically, in vitro tests are cell- or tissue-based experiments. The aim is to study the biochemical functions of the target as a result of binding to potential drug ligands. Parameters such as ionic concentrations, enzyme activities, and protein expression profiles are studied. 

Protein expression profiling (protein differential display) using microarrays is considered an important new tool for proteomic discovery. It is similar in concept and approach to the gene expression microarray for mRNA profiling. Sreekumar and Chinnaiyan (2002) describe a general approach for using the microarray to monitor protein expression in cancer and normal tissues. Here are the steps ... 

Protein Expression Profiling Pathways Drug Discovery ... 

Currendy, two types of fluorescent dyes are available fi om GE Heathcare (Table 1) (43, 45). We first reported that the use of an ultra highly sensitive fluorescent dye (GyDye DIGE Fluor saturation dye, GE Healthcare) enabled protein expression profiling even... 

To analyze the differenees of protein expression profiles between caneer tissues and corresponding non-caneerous tissues, the proteomic differential display method was used. In this method, 2-DE and MS were used to identify the proteins. We first separated proteins from caneer tissues and eorresponding non-cancerous tissues by 2-DE. Then the protein spots of the samples from eaneer tissues were compared to the spots of the samples from corresponding non-eaneerous tissues by using software for proteomie differential display. 

Shen J, Person MD, Zhu J et al. Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry. Cawcer Rex 2004 64 9018-9026. 

Jenkins, R.E., Pennington, S.R. (2001). Arrays for protein expression profiling, towards a viable alternative to two-dimensional gel electrophoresis Proteomics, 1(1), 13-29. 

A promising recent approach for understanding the biologic effects of biomaterials is the use of modern proteomics approaches. Such techniques have been used for the purpose of measuring protein expression profiles of macrophages (cultured on different biomaterials) to learn more about biocompatibility. Proteomics techniques were able to demonstrate that different types of polyurethane materials led to different intracellular and structural protein expression profiles.73... 

The ESI-LC/MS-based approaches that feature ion trap (Gatlin et al, 1998 Washburn et ah, 2001) and quadrupole time-of-flight (QTOF) (Blackburn and Moseley, 1999) mass spectrometers are routinely used for the identification and characterization of proteins. Nanoelectrospray LC/MS formats (Figure 6.3) are used to provide lower limits of detection and fully automated sample preconcentration and desalting. On-line LC/MS approaches for protein expression profiling are also used with ESI-TOF (Banks and Gulcicek, 1997 Chong et al., 2001) and ESI-Fourier transform (FT) (Kelleher... 

Quantitation Once protein expression profiling activities characterize qualitative features, the attention turns to delineating protein interactions and mechanistic pathways responsible for disease. These studies also require rapid sequence determination/confirmation combined with accurate and sensitive quantitative analysis. The quantitation approaches would allow for direct comparison of protein amounts (absolute or relative) from a variety of cellular states. Because of the reasons stated previously, quantitative applications are likely to be less dependent on 2-DGE and rely primarily on formats that involve specific purification and/or chromatographic separation with mass spectrometry. 

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