Protein quantification chemical labeling

Many analytical strategies have been developed for stable-isotope-based quantification of proteins, which all differ in the way stable isotopes are introduced into peptides or proteins (Fig. 15). They can be classified as (1) metabolic labeling, where cells acquire stable isotopes from the growth medium and incorporate them during protein biosynthesis , (2) enzymatic labeling, where stable isotopes are incorporated by an enzymatic reaction performed in vitro (3) chemical labeling, where stable isotopes are introduced by a chemical reaction in vitro and (4) spiking in a labeled reference compound. 

The isotope-coded affinity tag (ICAT) technique involves differential labeling of two different protein populations on the side chain of reduced cysteinyl residues using one of two chemically identical but isotopically different ICAT reagents (19). By incorporating a biotin affinity tag into the ICAT reagents, selective isolation and purification of labeled peptides substantially reduces sample complexity. The ICAT approach has been applied successfully to the systematic identification and quantification of proteins contained in the microsomal fraction of cells... 

One of the main problems to overcome in order to realise quantitative proteomics is that mass spectrometry is not strictly a quantitative technique with respect to molecular abundance. One approach to overcome this problem is to perform comparative experiments in parallel where one sample is labelled with an isotope (such as 0 or H) and the other is unlabelled. Mass differences between labelled and unlabelled samples may be observed in a mass spectrometer and the relative ratios quantified (Figure 9.21). Obviously this method of quantification relies on equivalent mass expression by both samples in spite of the isotopic labelling of the one and not the other. In other words, in spite of the labelling, both samples should behave identically chemically. The simplest way to effect labelling is to use isotope-coded affinity tags (ICATs). According to this technique, two identical populations of proteins... 

Isotope-coded affinity tag (ICAT) peptide labeling is an approach that combines accurate quantification and concurrent sequence identification of individual proteins in complex mixtures (Gygi, et al 1999). This method is based on a newly synthesized class of chemical reagents used in combination with tandem mass spectrometry. The method consists of four steps ... 

The contribution of MS to identification of compounds and quantification of their concentration is complementary to other detection techniques and, despite being very practical and versatile, it remains fundamentally replaceable. However, knowledge of molecular weight is a prerequisite for techniques that rely on the synergies with stable isotopic tracers. In fact, powerful analytical methods exist to obtain important insights on cell dynamics from the ratiometric measurement of marked and not-marked species (or atoms). We cite, for example, (1) relative abundances of virtually all metabolites or proteins in two separate cultures are quantified based on the isotope dilution theory [43 5] (2) information on the mechanisms and kinetics of nonlinear chemical processes can be extracted from response tracer experiments [46 7] and (3) the labeling patterns in metabolic intermediates are used to resolve the relative rate in convergent reactions in vivo [48,49]. 

The above facts do not favour protein or peptide quantitation using MALDI-MS. Some problems are associated with MALDI-MS quantification i) low shot-to-shot reproducibility, ii) various signal suppression effects, and iii) strong influence of sample preparation and matrix crystallisation. Nevertheless, it is possible to use MALDI-MS to obtain absolute or relative quantitation. In most cases, the idea is to use an internal standard for an absolute quantitation, but this standard must have the same physico-chemical characteristics as the quantified peptide. The use of a different peptide in terms of sequence may result in different desorption and ionisation properties. Usually, the internal standard is the same peptide labelled with a stable isotope to modify slightly the mass. 

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