A Metal-coded Affinity Tag Approach to Quantitative Proteomics

Abstract

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...

Inductively coupled plasma- and glow discharge plasma-sector field mass spectrometry : Part II. Applications

“…However, CYP2E1 levels appear to be modulated to some extent by the compounds So far, the previous applications have been related to the labelling of antibodies used for immune-reactions, but in the following example it should be demonstrated that this procedure can also be applied for proteins directly, as it was discussed in a review article recently. 370 Ahrends et al 371,372 used metal-coded affinity tags (MeCAT) for direct labelling of standard proteins and eye lens proteins for quantitative ICP-SFMS. As MeCAT reagents they chose derivatives of the lanthanide chelating DOTA to which a cysteine reactive maleimide moiety was attached via a spacer.…”

Inductively Coupled Plasma–Mass Spectrometry (ICP-MS) for Quantitative Analysis in Environmental and Life Sciences: A Review of Challenges, Solutions, and Trends

“…In terms of flexibility, labeling with lanthanides using bi-functional chelating agents, which are covalently bound to the targeted bio molecule, and which form highly stable complexes with the metal ion, represents the current state of the art within this field. [33][34][35] Lanthanides are ideal elements for such labeling approaches because they can be detected with high sensitivity via ICP-MS due to a negligible background, as well as the absence of interferences.…”

“…199 A similar approach has been recently used for the absolute quantification of iodinated peptides using capillary LC hyphenated to ICP-MS. 32 During the last few years labeling using lanthanides and bi-functional chelating agents gained much interest and also represents the latest trend in ICP-MS based quantitative analysis. 24,27 Their application for absolute protein quantification, 35 multiplexed immunohistochemical detection of tumor markers, 200 peptide quantification, 201 or multiplexed bioassays 202 indicate the future potential of such labeling approaches.…”

Accurate quantification of modified cyclic peptides without the need for authentic standards

“…[26][27] For these reasons it has become a significant and complementary technique in bioanalysis for the determination of biomolecules and quantification of therapeutic agents. [28][29][30][31][32][33][34][35][36] Application of ICPMS allows the quantification of elements independent of their molecular form, hence the analyte retains its original form during quantification. Coupled with molecular information obtained from ESI-MS or MALDI enables the compound identification simultaneously with its quantification.…”