Gel-Free Proteomic Approaches

In gel-free proteomic approaches, also named shotgun proteomics, the entire proteome of a biological system is enzymatically digested and the very complex peptide mixture thus obtained, containing thousands of peptides, is directly analyzed by two-or multi-dimensional LC-MS/MS 

Theoretical and technical aspects of mass spectrometry and proteomics have been extensively reviewed (Aebersold and Mann 2003 Walther and Mann 2010 Angel et al. 2012). 

Proteomics for Studying Moiecuiar Mechanisms of Probiotic Action 

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Advantages and Applications of Gel-Free Proteomic Approaches in the Study of Prokaryotes... 

The membrane-bound cytochrome P450 enzymes represent key players in secondary metabolite pathways as they catalyze vital, often rate-limiting steps. However, basic, hydro-phobic and membrane-spanning proteins remain an intrinsic limitation for 2D-PAGE [118]. Adoption of gel free proteomic approaches such as iTRAQ (Table 6) may help to overcome this bottleneck. 

The subsequent downstream processing section, which includes visualization of quantified proteins, statistical validation of differences in treatments or samples, and biological interpretation, is much less defined in terms of work-flow regimens and is discussed toward the end of this chapter. In the succeeding text, various relevant aspects of proteomic workflows that impinge on the data obtained from proteomic analysis of prokaryotes assuming that a gel-free approach is used are discussed. 

In the gel-free approaches, also referred as shotgun proteomics, proteins in a mixture are digested and the peptides released separated by reverse-phase HPLC, either alone or combined with strong cation exchange chromatography... 

A modified version of gel-free ABPP, introduced by Speers and Cravatt,29 overcomes these limitations by the application of a tandem orthogonal proteolysis (TOP) strategy for the parallel and full characterization of probe-labeled proteins and sites of modification. In this approach, proteomes were first labeled with a... 

In this chapter we will review proteomic investigations of cardiac proteins and focus on their application to the study of heart disease in the human and in animal models of cardiac dysfunction. The majority of these studies of the cardiac proteome have involved protein separation, visualisation and quantitation using the traditional 2-DE approach combined with protein identification by mass spectrometry. These essential technologies will be briefly described. However, there is increasing interest in using alternative gel-free techniques based on mass spectrometry or protein arrays for high throughput proteomics. These alternative approaches will be introduced, but further details can be found in Chapter 2 of this volume by Michel Faupel. 

Peptide sequence tags with multidimensional protein identification technology (MudPIT). MudPIT is a gel-free shotgun proteomic approach, which does... 

Since proteomics began with 2-DE methodology, the apphcation of MS has been driven by the qualitative character of protein identification on a 2-DE gel. Indeed, MS techniques are very convenient for protein identification. ITowever, their apphcation to protein quantification is more comphcated since there is no hnear dependence between the concentrations of protein or peptides in a sample and the MS signals observed. While there are several promising gel-free MS-based approaches, presently available methods do not fulfill the increasing need for rehable methods of absolute quantification of proteins (Sanz-Medel et al. 2008). 

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