Quantitative protein abundance comparison

If the purpose of gel electrophoresis is to identify low-abundance proteins (e.g., low-copy-number proteins in a cell extract or contaminants in a purification scheme), then a high protein load (0.1 to 1 mg/ml) and a high-sensitivity stain such as silver or fluorescence should be used. When the intention is to obtain enough protein for use as an antigen or for sequence analysis, then a high protein load should be applied to the gel and the proteins visualized with a staining procedure that does not fix the proteins in the gel, e.g., colloidal CBB G-250 (Subsection 8.2.8.1). Furthermore, for purposes of quantitative comparisons, stains with broad linear ranges of detection response should be used. 

Proteomic analysis frequently involves comparison of protein levels in experimental and control samples, which is linked with the quantitative analysis (see Chapter 8). In some cases, it is necessary to follow protein abundances over time. Changes in protein levels in cells or biofluids may be monitored over a few hours or days, which does not put much pressure on the temporal resolution of the analytical procedure [at least not to the extent observed in ultrafast time-resolved mass spectrometry (TRMS) studies introduced in other chapters of this book]. LC-MS is a simple approach to obtain relative abundances of proteins in highly complex samples [30]. It is possible to carry out quantitative comparisons... 

In order to profile proteins in the sample to confirm that changes occur in carbonylation stoichiometry rather than in the protein abundance. Scaffold allows for simultaneous comparison of multiple proteomic data sets in which the list of identified proteins can be sorted by various parameters. The spectral counting technique for relative protein quantitation utilizes the total number of MS/MS spectra identified for a particular protein as a measure of protein abundance, and consequently this parameter can be used to classify the abundance. Published protocols [14,15] can be used as methods for relative quantitation in which the change in abundance can be determined by the ratios as follows ... 

AQUA allows for direct comparison of proteomic data obtained on different instruments and in different laboratories. The need to measure the absolute abundance of proteins is achievable using a targeted quantitation approach. A synthetic, isotopically labeled peptide is used as the IS. This peptide is the isotopic counterpart of a signature peptide, which represents a 1 1 ratio... 

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