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. Proteomics as a field of research is based on the characterization of an entire proteome of a biological system. A variety of approaches have been developed during the last decades to characterize such mixtures of proteins and peptides, and necessarily, all of them use separation techniques. At the protein level, separation has been achieved using 2-D 1 gel electrophoresis (1) and densitometry of stained proteins or fluorescence detection (2). After digestion of the proteins, peptides were identified using liquid chromatography, mass spectrometry, or both (3, 4). However, this information was only qualitative. It became rapidly evident that quantitative data were definitively required, e.g. for the characterization of dynamic biological systems or the search for biomarkers in clinical proteomics. Subsequently methods have been developed for the quantitative determination of proteins and peptides mainly based on chemical or metabolic isotopic labeling combined with LC/MS n detection (5, 6). Label-free LC/MS quantitative strategies are under development as well (7).Using such techniques, the investigation of changes of the proteome in biological systems has become possible. However, o...

Group II introns are large, natural catalytic RNAs or ribozymes that were discovered in organelles of certain protists, fungi, algae, and plants and more recently also in prokaryotic organisms. In vitro, some members were found to self-splice from their pre-RNAs by two consecutive transesterification reactions joining the flanking exons and releasing the intron in a typical lariat form. Apart from self-splicing, a variety of other in vitro activities have been detected for group II introns demonstrating their amazing catalytic versatility. Group II introns fold into a conserved secondary structure consisting of six domains radiating from a central wheel that brings the 5' and 3' splice junction into close proximity. Domain 1 is the largest domain that is assumed to deliver the molecular scaffold assembling the intron in its active structure, while domain 5 is the phylogenetically most conserved part that represents the active site of the ribozyme. In vivo, the splicing reaction of many, if not all group II introns is assisted by proteins either encoded by the introns themselves (maturases), or encoded by other genes of the host organisms. The host proteins known to date have additional cellular functions and seem to have been adapted for splicing during evolution. Some of the protein-encoding group II introns were also shown to act as mobile genetic elements. They can integrate efficiently into intronless alleles of the same gene (homing) and at much lower frequencies into ectopic sites (transposition). The mobility process depends on intron encoded protein functions (endonuclease and reverse transcriptase) and on the intron RNA. This review provides a comprehensive survey of the structure/function relationships and the reaction potential of group II introns, the structurally most complicated, but also most fascinating ribozymes when looking at their catalytic repertoire in vitro and in vivo.

Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.

A method for the analysis of Pt-protein complexes in biological samples, previously subjected to cisplatin treatment, has been developed. Proteins were separated by gel electrophoresis, and those bound to Pt were detected with high sensitivity by LA-ICP-(SF)-MS. Pt-containing spots were in-gel digested with trypsin, and the peptides produced identified using nHPLC-ESI-LTQ-FT-MS/MS. The influence of protein separation conditions, staining and gel processing prior to laser ablation on Pt-protein bonds preservation have been evaluated using standard proteins incubated with cisplatin. 2-DE separation under non-reducing conditions followed by either Coomassie blue brilliant or silver staining is appropriate for Pt-protein complexes, achieving a good separating resolution of the proteins in biological samples. Direct LA-ICP-MS analysis of glycerol-treated dried gels for Pt-protein monitoring resulted in better sensitivity, more reliable relative Pt signals and a simpler and less timeconsuming approach compared to the analysis of blotted membranes. Ablation of gels allowed tackling protein identification of Pt-spots in the remaining non-ablated material in the gel, making it unnecessary to run several gels in parallel for separate Pt detection and protein identification. By using this approach, Pt coordinated to proteins, such as a-2-macroglobulin, transferrin, albumin or hemoglobin, was detected in the serum from a rat treated in vivo with cisplatin after nrSDS-PAGE separation. Furthermore, the first complete LA-ICP-MS metalloprotein contour map in a 2-DE gel has been produced, in this case for the detection of Pt-protein complexes in renal proximal tubule epithelial cells (RPTECs) incubated with cisplatin. Several proteins were identified in those spots containing Pt, which may have a connection with the drug-induced nephrotoxicity mainly affecting this cell type in the kidney.

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