Proteomics bottom

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. 

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. 

Millea, K.M., Krull, I.S., Cohen, S.A., Gebler, J.C., Berger, S.J. (2006). Integration of multidimensional chromatographic protein separations with a combined top-down and bottom-up proteomic strategy. J. Proteome. Res. 5, 135-146. 

The enzymes used for bottom-up proteomic studies can be classified as those with specific cleavage specificity and those with nonspecific proteolytic activity. 

FIGURE 15.2 Common protein ionization methods used for MS-based proteomics. Two common ionization technologies are currently available for protein analysis. Top ESI volatilizes and ionizes peptides and proteins in solution. Bottom MALDI uses analytes that are co-crystallized in a matrix composed of organic acid on a solid support. A pulse of ultraviolet laser evaporates the matrix and analyte into gas phase, resulting in generation of single charge ions. 

B. Bogdanov and R. D. Smith. Proteomics by FTICR Mass Spectrometry Top Down and Bottom Up. Mass Spectrom. Rev., 24(2005) 168-200. 

Several approaches are utilized to study systems biology. The bottom-up approach starts from the molecular level, the omics, to identify and evaluate the genomic and proteomic basis of diseases. The top-down approach attempts to integrate human physiology and diseases to provide models to understand disease pathways at organ levels. 

Figure 1.10 Top Genome-based arrays. Bottom Proteome-based arrays.

Bogdanov, B., and Smith, R. D. (2005). Proteomics by FTICR mass spectrometry Top down and bottom up. Mass Spectrom. Rev. 24 168-200. 

Carbonylation occurs by the oxidation of some amino acid side chains into ketone or aldehyde derivatives by reactions with compounds of lipid oxidation or by glycoxidation with reducing sugars. These protein-carbonyl compounds are markers of protein oxidation, and recently, several carbonylated proteins and protein oxidation sites in milk (96), meat (97), and fishes (98) have been identified using a classical bottom-up proteomics approach based on 2-DE and MS/MS. Specific labeling of protein carbonyls using fluorescein-5-thiosemicarbazide has been developed and combined with 2-DE and... 

The study of proteins is especially interesting in food safety because they may act as toxins, antinutrients, or allergens (53). Proteomics, a high-throughput technology able to quantify hundreds of proteins simultaneously, has become very important in comparative studies of GM plants and their nonmodified counterparts (54). Two conceptually different strategies can be followed in comparative proteomics the shotgun and the bottom-up approaches. 

The use and development of high-resolving separation techniques as well as highly accurate mass spectrometers is nowadays essential to solve the proteome complexity. Currently, more than a single electrophoretic or chromatographic step is used to separate the thousands of proteins found in a biological sample. This separation step is followed by analysis of the isolated proteins (or peptides) by mass spectrometry (MS) via the so-called soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) combined with the everyday more powerful mass spectrometers. Two fundamental analytical strategies can be employed the bottom-up and the top-down approach. 

A growing number of researchers are focusing on the use of top-down proteomics, a relatively new approach compared to bottom-up, in which structure of proteins is studied through measurement of their intact mass followed by direct ion dissociation in the gas phase. The main advantages over the bottom-up approach are that higher sequence coverage is obtained, it permits... 

Figure 5.2 LC workflow strategy options in proteomics. a Bottom-up approach, b Top-down approach.

The same concept in proteomics studies has technological implications, e.g., which method, sample preparation protocols, and instrumentation will be used. Again, top-down analysis will be based on isolation, analysis, and characterization of an intact protein to reveal its function. Fourier transformed ion cyclotron resonance mass spectrometry (FT-ICR) (Marshall et al., 1998) facilitates such approach in protein identification as a result of random fragmentation of an intact molecule. In contrary, bottom-up approach is based on up-front fragmentation of the protein in question using various proteolytic enzymes with known specificity (Chalmers et al., 2005 Millea et al., 2006). In these experiments, trypsin is most commonly used. An important question that remains is whether more... 

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