Reproducibility protein profiling

Variations on the spectral peaks from different species of the same genus were also observed. Three species of Pseudomonas produced the spectra shown in Figure 14.2. These spectra are clearly unique and were used to correctly identify unknown samples. Because of peak ratio reproducibility issues in bacterial protein profiles obtained by MALDI MS,11 a fingerprint approach that had been used for other mass spectrometry approaches has not been used. The profile reproducibility problem was first recognized by Reilly et al.12,13 and later researched by others in the field.14,15 As a later alternative, a direct comparison of the mass-to-charge ratio (m/z) of the unknown mass spectral peaks with a database of known protein masses has been used to identify unknown samples.14... 

The reproducible high-resolution separation of protein mixtures is the main purpose of proteome analysis. O FarelTs classic tube gel technique has limited reproducibility. It is often difficult to compare the protein profiles obtained using O FarelTs method in different laboratories. In some cases, the data obtained even in the same laboratory by different operators are not comparable. 

Albrethsen, J. 2007. Reproducibility in protein profiling by MALDI TOP mass spectrometry. Clinical Chemistry 53 852-858. 

The other approach to quantitative protein profiling is based on stable isotope labelling of proteins and peptides and automated tandem mass spectrometiy (MS/MS) (Gygi et al., 1999b). This method, termed isotope-coded affinity tag labelling or ICAT, has been shown to be robust, reproducible and amenable to high throughput automation (Ideker et al.,... 

Figure 7 Protein profiles of virally infected cells. Two-dimensional protein profiles of rhabdomyosarcoma cells (A) uninfected and (B) infected with Cocksackie B5 virus. The proteins were labeled and located by autoradiography. The three proteins p39, p54, and p75 are specific for the infected cells. (Reproduced with permission from Argo E, Gimenez B, and Cash P (1992) Protein profiles of virally infected cells. Archives of Virology 126 215-229 Vienna Springer-Verlag.)...

Bacteria cell proteins constitute another level of genomic expression. The amino acid sequence of proteins is the result of a direct translation of genes. It is therefore normal to distinguish bacteria from one another by the proteins that they contain. The primary structure determines molecule mobility in an electrophoretic gel in conditions where the secondary, tertiary and quaternary structures are denatured. This identification method therefore involves subjecting the total cell contents of bacteria to electrophoresis. After staining, the protein profiles are compared either visually or by computer-assisted analysis. The electrophoretic profiles are reproducible. They are standardized by markers which are required to compare several gels. 

An investigation of several methodological aspects of obtaining urinary protein profiles by SELDI-TOF-MS revealed that among the extrinsic factors instrument settings and matrix composition critically influenced peak detection and reproducibility, while freeze-thaw cycles had minimal effects. Intrinsic factors of significance included blood in urine, dilution, and first-void vs. midstream urine [100]. 

Peptide map analysis of a protein containing two or more cysteine residues typically employs reduction and alkylation chemistry for efficient, reliable proteolysis, reproducible chromatographic profiles, and straightforward characterization by MS. However, when protease digestion is carried out on a nonreduced protein, the disulfide bonds in the protein will maintain the covalent linkage between the peptides that are involved in the disulfide bond. In many cases, it is then possible to choose a protease that will ensure one cysteine residue in each peptide upon cleavage, such that each disulfide bond will associate with a pair of proteolytic peptides. 

Figure 5.19 MALDI-ToF mass spectrum, providing a molecular-weight profile of the tryptic peptides derived from spot 22 (see Figure 5.18) of the silver-stained two-dimensional gel of the proteins extracted from the yeast S. cerevisiae. From Poutanen, M., Salusjarvi, L., Ruohonen, L., Penttila, M. and KaUddnen, N., Rapid Commun. Mass Spectrom., 15, 1685-1692, copyright 2001. John Wiley Sons Limited. Reproduced with permission.

Two features of expression profiling make it the most productive approach to study biological systems for the immediate future. First, the present efficiency with which investigators can obtain global and quantitative information with DNA arrays exceeds that of proteomic techniques. Second, RNA expression profiles provide an extremely precise and reproducible signature of the state of the cell that probably reflects albeit indirectly, the functional state of all proteins (Young, 2000, p. 13)-... 

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