Protein expression, differential

Figure 3.2. Stable isotope labeling for quantifying differential protein expression. Cell populations are grown in either 14N or 15N containing medium. Protein lysates are fractionated and separated by 2D gel electrophoresis. Protein spots are excised, digested with trypsin and the mass of the resulting peptides is determined by mass spectrometry. The presence of 15N results in a shift and creates two peaks for each peptide. The ratio of intensities of the peaks is indicative of the relative expression levels of the proteins. Spot A contains a protein that is expressed at similar levels in both cell pools. Spot B contains a protein that is expressed at higher levels in cell pool 2. Figure adapted from Oda et al. (1999).

Chevalier S et al. Proteomic analysis of differential protein expression in primary hepatocytes induced by EGF, tumour necrosis factor alpha or the peroxisome pro-liferator nafenopin. Eur J Biochem 2000 267 4624-4634. 

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. 

IFl-3). In contrast, eukaryotic initiation is a rather complex process involving a large number of initiation factors (elFs, Table 1). This is also the stage of eukaryotic ribosomal protein synthesis, which is most highly regulated to achieve differential protein expression. Elaborating the details of eukaryotic initiation is beyond the scope of this chapter. 

C Vasseur, J Labadie, M Elebraud. Differential protein expression by Pseudomonas fragi submitted to various stresses. Electrophoresis 20 2204-2213, 1999. 

FIGURE 7 Group B streptococcus infection-induced differential protein expression in nonhuman primate (A) and human (B) amniotic fluid samples by surface-enhanced laser desorption/ionization (SELDI-TOF MS) using normal-phase protein chip arrays. Spectrum from 2.5 to 15 kDa collected at 235 nm laser intensity. Detailed spectra show increased expression of the 3.5 and 10.8 kDa peaks between control and infected. Arrows indicate the unique peaks represented by polypeptides overexpressed in infection. 

General consensus has sub-divided proteomics into three main areas, Expression Proteomics, Functional Proteomics, and Structural Proteomics. Expression Proteomics (sometimes called differential-expression proteomics) involves the analysis of differential protein expression by protein... 

Lexander H, Franzen B, Hirschberg D, Becker S, Hellstrom M, Bergman T, et al. Differential protein expression in anatomical zones of the prostate. Proteomics 2005 5(10)2570-2576. 

Hathout, Y, Riordan, K., Gehrmann, M., Fenselau, C. (2002). Differential protein expression in the cytosol fraction of an MCF-7 breast cancer cell line selected for resistance toward melphalan. 

Normal blood vessels are made up of many different cell types, including smooth muscle cells, endothelial cells, and fibroblasts. The situation is even more complex in atherosclerotic vessels due to the inflammatory nature of the disease, so that cell types, such as T-lymphocytes and macrophages, are also present in large numbers. Thus, proteomic analysis of intact vessels, particularly if the study is designed to investigate differential protein expression in diseased and normal vessels, is very challenging. 

To cope with the high number of RP-HPLC runs required to analyze fractions in the two-dimensional LC-MS approach, multiplexed LC-separation and spotting for LC-MALDI analyses was applied very early. In quantitative analysis of differential protein expression by ICAT technology, total analysis time could be significantly reduced with four parallel separation lines (Lee et al. 2002). Prerequisite is of course... 

Before any additive is to be used with a given cell type, it should first be tested to ensure that it has no detrimental effects on cell growth, metabolism, differentiation, protein expression, etc., and that it does indeed offer mechanical protection in agitated and/or aerated systems (bioreactors). 

Microfluidic devices that will enable global proteomic profiling will be particularly useful for biomarker discovery applications where the researcher is interested in comprehensively mapping all peptide/protein components in given biological fluids or tissues. Differential protein expression analysis will enable the discovery of specific protein markers or protein co-expression patterns that... 

Figure 1. Use of 2D gels to study differential protein expression in the parasite Leishmania. Examples inclnde the identification of dmg-resistance proteins (green), strain or species-specific markers (bine), stage-specific proteins (orange), and the effects of host-parasite interactions on both the parasite (teal) and the host (violet).

Prokai, L., Stevens, S.M. Jr., Rauniyar, N., Nguyen, V. (2009) Rapid label-free identification of estrogen-induced differential protein expression in vivo from mouse brain and uterine tissue. Journal of Proteome Research, 8, 3862-3871. 

Two studies have used LC/MS/MS to identify differential protein expression in HIV- or HCV-infected cells. In the first study, traditional HPLC (ion exchange and reverse-phase columns) coupled to an ultrasensitive ion trap MS was employed to... 

MS imaging has also been performed on many of these tissues to serve as a visual comparative analysis of differential protein expression. For example. Fig. 5... 

Growth factors are involved in cell division, migration, and differentiation, protein expression, and enzyme prodnction. The wound-healing properties of growth factors... 

Fig. 16 The ICAT strategy for quantifying differential protein expression, a Structure of the ICAT reagent, b Schematic of the ICAT strategy. Reproduced from [10] with permission from Birkhauser Verlag, 2006...

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