Protein expression, quantitation

In this volume not all stress types are treated. Various aspects have been reviewed recently by various authors e.g. The effects of oxygen on recombinant protein expression by Konz et al. [2]. The Mechanisms by which bacterial cells respond to pH was considered in a Symposium in 1999 [3] and solvent effects were reviewed by de Bont in the article Solvent-tolerant bacteria in biocatalysis [4]. Therefore, these aspects are not considered in this volume. Influence of fluid dynamical stresses on micro-organism, animal and plant cells are in center of interest in this volume. In chapter 2, H.-J. Henzler discusses the quantitative evaluation of fluid dynamical stresses in various type of reactors with different methods based on investigations performed on laboratory an pilot plant scales. S. S. Yim and A. Shamlou give a general review on the effects of fluid dynamical and mechanical stresses on micro-organisms and bio-polymers in chapter 3. G. Ketzmer describes the effects of shear stress on adherent cells in chapter 4. Finally, in chapter 5, P. Kieran considers the influence of stress on plant cells. 

The Gmuender (2001) study also illustrates the limitations in terms of the quantitative reproducibility of protein expression mapping using 2D gels. They found that 32% of the spots on the 2D gels exhibited greater than... 

The use of the in vivo labeling methods described above is limited by the fact that the sample must be grown in the presence of the labeling isotopes. In many cases, it is not feasible to perform in vivo metabolic labeling. For example, for human clinical samples it is not possible to perform in vivo labeling and yet it is highly desirable to obtain accurate quantitative information on protein expression levels within these samples. Therefore, robust methods are needed for quantitation of protein levels in the absence of in vivo labeling with isotopes. 

Oda, Y., Huang, K., Cross, F. R., Cowbum, D., and Chait, B. T. (1999). Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591-6596. 

Goodacre, R. Karim, A. Kaderbhai, M. A. Kell, D. B. Rapid and quantitative analysis of recombinant protein expression using pyrolysis mass spectrometry and artificial neural networks Application to mammalian cytochrome b5 in Escherichia coli. J. Biotechnol. 1994,34,185-193. 

The feasibility of this approach to not only differentiate pathogenic and nonpathogenic strains of bacteria based on significant differences in protein mass but also on the basis of variations in levels of protein expression was demonstrated using a method for quantitating protein expression by LC/MS of whole proteins.54 This method is based on the fact that some proteins present in cells are abundant universal proteins whose expression levels exhibit little variation. This method demonstrates that these co-extracted proteins can be used as internal standards to which the other proteins in the sample can be compared. By comparing the intensities of a selected protein to a marker protein, or internal standard, a relative ratio is obtained. This ratio... 

Kahn TW, Beachy RN, Falk MM (1997) Cell-free expression of a GFP fusion protein allows quantitation in vitro and in vivo. Curr Biol 7 R207-R208... 

Proteome profiling allows quantitative and global examination of the changes in protein expression in different strains or cells under different conditions. Two-dimensional gel... 

Cregger M, Berger AJ, Rimm DL. Immunohistochemistry and quantitative analysis of protein expression. Arch. Pathol. Lab. Med. 2006 130 1026-1030. 

Nirmalan NJ, Harnden P, Selby PJ, et al. Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting. J. Pathol. 2009 217 497-506. 

Isobaric labels thus permit quantitative information regarding protein expression levels in multiple samples analyzed simultaneously by MS. The multiplexed capability of these reagents allows the measurement of peptides and proteins in diseased samples, treated samples, and normal samples all in the same experiment. In addition, since all peptides from a given protein get labeled at their N-termini, the MS analysis generates more than one peptide signal, which can be used to confirm protein identity with greater confidence than using a cysteine label, like ICAT. 

The first step in biopharmaceutical development is the selection of a clone in a specific cell line. Whole-mass analysis, if possible, is a fairly simple and powerful tool at this stage to verify the successful expression and translation of the desired protein. VanAdrichem et al.65 described the use of MALDI MS to monitor protein expression in several mammalian cell lines like CHO DXB11, CHO SSF3, and hybridomas. Quantitative MALDI-TOF MS measurements of an IgG antibody and insulin during large-scale production in hybridoma cells were comparable to affinity chromatography results. 

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