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Molecular & Cellular Proteomics 6:1907-1916, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

A Metal-coded Affinity Tag Approach to Quantitative Proteomics^*

Robert Ahrends^,,¶, Stefan Pieper^,¶, Andreas Kühn, Hardy Weisshoff, Meike Hamester^||, Torsten Lindemann^||, Christian Scheler, Karola Lehmann, Kerstin Taubner^** and Michael W. Linscheid^,

From the Department of Chemistry, Humboldt-Universitaet zu Berlin, Brook-Taylor Str. 2, 12489 Berlin, Germany, ^|| Thermo Fisher Scientific, Hanna-Kunath-Str. 11, 28199 Bremen, Germany, ^** Institute for Analytical Sciences (ISAS), Albert-Einstein-Str. 9, 12489, Berlin, Germany, and Proteome Factory AG, Dorotheenstr. 94, 10117 Berlin, Germany

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.

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