Absolute quantitation peptides

Both absolute quantitation and relative quantitation of species in mixtures is of interest in some circumstances. Quantitation in a 5-minute analysis can be achieved by addition of an internal standard, ideally the target microorganism grown in special media to incorporate heavy isotopes92-95 and determination of the relative peak heights of pairs of proteins from the analyte and the standard. Isotope-labeled proteins or peptides, selected to match proteins or peptides characteristic of target microorganisms, can also serve as internal standards for isotope ratio measurement. The addition of unmatched proteins or peptides is less reliable for either ESI or MALDI measurements because of unpredictable suppression in the variable mixture. 

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. 

Isobaric Tags for Relative and Absolute Quantitation represents recent technology advancement, where four specific reagents label peptides in up to four samples. While the labeled peptides have the same mass, each label fragments into a different sized, characteristic tag that can be identified, and quantified in the tandem mass spectra In Arabidopsis iTRAQ has been used to map the organellarproteomes [123]. Another study applied iTRAQ to describe early changes to the phosphoproteome oiArabidopsis during defense response [124]. 

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