Quantitative peptide and protein analysis is one of the most promising fields in modern life science. Besides stable isotope coded labeling, metal chelate complexes are an alternative tool for quantification. The development of metal-coded affinity tags (MeCAT) was aimed to provide a robust tool for the quantification of peptides and proteins by utilizing lanthanide-harboring metal tags. It was shown that MeCAT is suited for relative quantification of proteins via standard mass spectrometric methods. The approach of tagging biomolecules with MeCAT offers the unique advantage of absolute quantification via inductively coupled plasma mass spectrometry (ICPMS), a well-established technique for assessing concentrations down to low attomole ranges. This work investigates the compatibility of MeCAT labeling to analysis workflows such as nano liquid chromatography/electrospray ionization tandem mass spectrometry (nano-LC/ESI-MS(n)). Focus was given toward the separation behavior of labeled peptides and the dynamic range of detection and peptide charge distribution. Furthermore, the stability of MeCAT under harsh analytical conditions was investigated. With the application of the MeCAT technique to a standard analysis scheme in proteomics, such as the investigation of changes in an Escherichia coli proteome, we successfully addressed the suitability to utilize MeCAT on biological samples. Furthermore, we demonstrated that MeCAT complexes are stable under a variety of conditions and that by applying LC/ESI-MS it is possible to cover a dynamic range of 2 orders of magnitude down to the low femtomole range with an average standard deviation below 15%. Therefore, this technique is suitable to common proteomic workflows and enables relative as well as absolute differential peptide quantification.

As the quantification of peptides and proteins extends from comparative analyses to the determination of actual amounts, methodologies for absolute protein quantification are desirable. Metal-coded affinity tags (MeCAT) are chemical labels for peptides and proteins with a lanthanide-bearing chelator as a core. This modification of analytes with non-naturally occurring heteroelements adds the analytical possibilities of inductively coupled plasma mass spectrometry (ICPMS) to quantitative proteomics. We here present the absolute quantification of recombinantly expressed aprotinin out of its host cell protein background using two independent MeCAT methodologies. A bottom-up strategy employs labeling of primary amino groups on peptide level. Synthetic peptides with a MeCAT label which are externally quantified by flow injection analysis (FIA)-ICPMS serve as internal standard in nanoHPLC-ESI-MS/MS. In the top-down approach, protein is labeled on cysteine residues and separated by two-dimensional gel electrophoresis. Flow injection analysis of dissolved gel spots by ICPMS yields the individual protein amount via its lanthanide label content. The enzymatic determination of the fusion protein via its β-galactosidase activity found 8.3 and 9.8 ng/μg (nanogram fusion protein per microgram sample) for batches 1 and 2, respectively. Using MeCAT values of 4.0 and 5.4 ng/μg are obtained for top-down analysis, while 14.5 and 15.9 ng/μg were found in the bottom-up analysis.

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Elevated cardiovascular risk in postmenopausal women and beneficial actions of estrogen replacement in animal models have been related to protective effects of estrogens. However, randomized trials of hormone replacement therapy with synthetic estrogens in humans failed confirmation and phytoestrogens, natural plant hormones with agonistic properties for estrogen receptors, could represent potential alternatives. The aim of the present study is to characterize an animal model for alternative hormone replacement with genistein as a natural estrogenic compound. We performed a 2-DE/ESI-LC-MS approach in order to identify protein species varying with genistein receipt and sex in their relative abundance in the healthy murine heart (http://www.mpiib-berlin.mpg.de/2D-PAGE). Oral genistein treatment revealed a substantial effect on the relative abundance of both estrogen receptors. Several enzymes of the fatty acid metabolism and their transcriptional regulators varied differentially in male and in female animals, at the transcript and/or the protein species level. Increased levels of enzyme species involved in the oxidative phosphorylation and generation of ROS were accompanied by decreased amounts of antioxidants in male mice receiving genistein compared with control males, which have been previously associated with various pathological conditions. Exposure of female animals to genistein provoked an increased abundance of two species of LIM domain-binding protein and one species of desmin. These proteins have been associated with cardiac hypertrophy and our data warrant caution for the use of them as molecular markers, since the animals did not exhibit any histological signs of cardiac hypertrophy.