A new 2D-LC approach for quantitative proteomics u.. (MeTag)
A new 2D-LC approach for quantitative proteomics using MeCAT labeling and multidimensional chromatography coupled to ultra-high-resolution FT-MS-MS and ICP-MS
Start date: May 4, 2009,
End date: May 3, 2011
The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, several approaches have been devised in recent years, which generally allow only the relative quantitative analysis of peptides and proteins. We have presented proof of concept of a new MeCAT technique, which offers a quantitative determination of proteins and peptides. A macrocyclic metal chelate complex (DOTA) loaded with different lanthanides (Me(III)-ions) is the central part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids (here: Cysteine) is a reagent that permits to quantify peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection ICP-MS. With ICP-MS the metals can be detected with high precision, low detection limits and great dynamic range. The course of the labelling reaction has been studied using SDS-PAGE and MALDI-TOF-MS, ESI-MS and ICP-MS. To limit the width in isotopic spread and to increase the sensitivity, we used up to now the mono-isotopic lanthanides. The detection limit for Bovine Serum Albumin was calculated to 110 attomol; more examples have been analyzed. In this new project, 2D LC techniques interfaced to ESIMS and ICPMS will be combined with our metal tagging. Different LC types will be examined as 1st dimension and different 2D separation techniques as well. We assume that the detection limits for modified Proteins and peptides can be improved using the strategies similar to shotgun and Mudpit techniques; several model systems are available. In the next step, MeCAT will be applied for the analysis of bacterial proteomes and membrane proteins. In addition new concepts for antibody tagging will developed fro the specific detection of antigens in target proteins of medical significance.
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