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...
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.
Quantitative proteomics has become an important method in modern life sciences. Besides protein identification, the aspect of quantification is of rapidly increasing relevance. MeCAT (metal-coded affinity tagging) is able to provide a tool that enables relative as well as absolute quantification. For structural elucidation, knowledge on the fragmentation behavior of MeCAT-modified peptides is highly beneficial. Therefore, the fragmentation behavior of MeCAT-labeled peptides under collision induced dissociation (CID), electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) conditions was studied. Application of CID and ECD allowed a straight-forward sequence elucidation of MeCAT-labeled peptides. During IRMPD all MeCAT-labeled peptides form characteristic fragments resulting from the fragmentational cleavage of the tagging group, thus, providing a screening method for the identification of labeled compounds. Furthermore, occurring side reactions during the labeling process were investigated. By-products were structurally characterized and reaction conditions were optimized in order to prevent the formation of these.
Reversible chemistry, allowing for chain-forming as well as chain-breaking steps, is important for biological self-organization. In this context, ribozymes, catalyzing both RNA cleavage and ligation, may have significantly contributed to extending the sequence space and length of RNA molecules in early life forms. Here we present an engineered RNA that self-processes by passing through a number of cleavage and ligation steps. Intermolecular reactions compete with intramolecular reactions, resulting in a variety of products. Our results demonstrate that RNA can undergo self-oligomerization, which may have been important for extending the RNA genome size in RNA world scenarios.
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