ELEMENTAG · A new concept for protein quantification by heteroatom tags

Final Activity Report Summary - ELEMENTAG (A new concept for protein quantification by heteroatom tags)

Proteins and peptides are an essential part of all living systems from the most elementary to the most complex. They are concerned with all physiological processes and in this connection they are of considerable interest in biochemical and clinical research. Proteomics is the systematic study of the many and diverse properties of proteins in a parallel manner, with the aim of providing detailed descriptions of the structure, function and control of biological systems in health and disease. Currently, the biggest unmet need in protein analysis is a method for the quantitative measurement of proteins, an important prerequisite for an elucidation of protein expression and function with diagnostic and therapeutic relevance. In contrast to existing approaches based on molecular mass spectrometry, such as electrospray ionisation mass spectrometry (ESI-MS) and matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS), in this project new peptide and protein quantification methods were developed based on metal tags in combination with element mass spectrometry (ICP-MS). This had the potential to open up entirely new possibilities for sensitive and accurate protein quantification. However, the development of metal tags especially designed for proteins and peptides analysis by ICP-MS remained so far still in its infancy. The most important scientific achievement was the highly specific labelling of peptides, peptide mixtures and protein digests with the rare earth metal lutetium in form of a metal complex with the chelator diethylenetriamine pentaacetic acid (DTPA). Labelled peptides were separated on a chromatographic system and specifically detected on the metal signal by ICP-MS. This method was highly sensitive and allowed for the detection of peptides at the attomole level, which corresponded to 10 to 18 mol, a milliardth of a milliardth mol, and was 10 000 times more sensitive in comparison to a method that was utilised before in the same laboratory. The chromatographic separation system was miniaturised so that the analysis could be performed in only 10 nanolitres, i.e. 10 milliardths of a litre, of sample. By calibration of the system with a stable isotope of lutetium (176Lu), that is by isotope dilution analysis, peptides could be quantified with the high precision of 4.9 %. Furthermore, different metal chelators such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were investigated and methods for peptide labelling were optimised. The methods developed were applied to insulin, a small protein with diagnostic and therapeutic relevance. In general, metal labelling had the potential to become a generic approach for highly sensitive absolute quantification of biomolecules by element MS, independent of whether detectable elements were naturally incorporated or not.

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