Development of arginine selective analysis principles

Peptides and proteins containing two or more adjacent arginine (Arg) residues as sequence characteristics can enter cells via an active transport mechnanism called "twin arginine translocation" (Tat) pathway. An important example of this Tat pathway is the nuclear transcription activator protein encoded by HIV type 1 that is required for viral replication, whereby arginines are known to serve as anion binding sites in the binding region. This (Arg)x facilitated transport mechanism and principle through the cell membrane has recently been reported as very useful active handle to be implemented also as drug transporter. This route is now considered as a more universal possibility which may enhance bioavailability of non-peptide but also of peptide like drugs. However, this principle is also responsible for the transport and affinity of many endogenous and exogenous peptides and toxin like compounds, so it deserves great attention also from an analytical point of view. The prime focus of the present project refers to the development of novel analytical tools and methods for selective enrichment, separation, identification and quantification of Arg containing bioactive compounds. This will be carried out by means of combined liquid chromatographic and mass spectrometric techniques hyphenated with Arg affine sorption and/or tagging principles. The present project will give a significant input to the developement of (i) multi-Arg (Arg)x but also for (ii) single-Arg containing peptides thus offering novel tools in proteomics research enabling the selective identification of arginine and/or guanidine containing entities in complex mixtures. The aim of one project pathway is the introduction of Arg-targeted selectivity principles to separation protocols to "fish out" arginine residues from a multi-component matrix (e.g. out of a tryptic digest) thus reducing significantly the complexicity of the matrix. This concept will enable also a strong analytes enrichment possibility. Parallel to the adsorption strategies main focus will be given to the selective non-covalent and covalent tagging of the arginine/guanidine residue to enhance MS/MS identification and to support also quantification. Accordingly the introduction of cold labelled Arg selective tags will be developed to offer a method to be used in the detection of up and down regulation of biomolecules in the course of the Life Sciences related events. The underlying principles of Arg selectivity are based on molecular and functional group recognition using multidimensional concepts.

The aim of the FWF project, entitled "Development of arginine selective analysis principles," centered on the elucidation of molecular recognition phenomena; involving the formation of selective covalently and non- covalently bound derivatives and adducts between the guanidinium group of the arginine side chain and suitable reaction partners. This lead to three main working strategies: (a) the development of chemoselective tagging and labeling concepts of the guanidinium group leading to products being well and uniquely detected by mass spectrometry (MS); (b) the transformation of this derivatization concept to the development of chemoselective "adsorption materials" for isolation of arginine-containing peptides out of a complex matrix of diverse peptides resulting from a protein digest; and (c) the elucidation of molecular recognition principles for molecular associates formed via intermolecular interactions, including those between the basic guanidinium group and the oxonium groups of various acids. The latter ESI-MS/MS related project aspect lead to a fast screening concept for the elucidation of stereoselectively- driven complexes and adducts in solution; with the advantage of providing qualitative and quantitative data regarding selectivity and binding strength of the interacting species. The scientific accomplishments achieved by many excellent co-workers are summarized in 21 publications in peer-reviewed journals. Some aspects of the research concepts were also filed as a granted EU patent.