Universally high throughput Mass spectrometry-Tag Quantification in biological matrices: Alternative to radiolabeling – MTaQ

Measuring the affinity of a ligand for its target is an issue of central importance in life science research and drug development. Since the affinity of reference ligand for target receptor is generally high, measurement of signal requires a very good sensitivity. Mass spectrometry offers sensitive and selective methodologies to quantify molecules in complex biological systems. Developing a sensitive MS-based universal approach for the quantification of tagged reference ligands in competitive binding assays will provide a breakthrough. The novelty of such an approach thus lies in the joint use of mass spectrometry and original labeling chemistries. Several robust molecular and elemental mass spectrometry technologies (ESI-MS, MALDI-MS, ICP-MS), which are available in the consortium gathered for the ANR MTaQ projet, are investigated in conjunction with different types of new chemical tags designed to improve the limit of detection and quantification. The project has started with a model receptor/ligand system (Vasopressin V1A receptor /AVP). The labeling of the AVP peptide ligand by an organic moiety aimed at specifically increasing the ionization in MALDI mass spectrometry of the tagged peptide present in a complex biological matrix (cell culture) constituted the first research topic by molecular mass spectrometry according to the scheduled tasks. In parallel, the tagging of the AVP peptide ligand by one or multiple selenium atoms was chosen to develop the second approach using elemental mass spectrometry (ICP-MS).

The peptides of the studied receptor/ligand system V1a/AVP (reference ligand AVP that is an agonist cyclic peptide, and HO-LVA, an antagonist linear peptide) were both successfully prepared and tagged by fixing the labeling moiety at the N-terminal end of the peptide sequence or by inserting a non naturel amino acid that contained the label. The available quantities and the high level of purity of the prepared samples allowed tackling the mass spectrometry and pharmacology tasks within the first year of the research project as scheduled. The affinity of all newly prepared tagged peptides for the V1a receptor was first checked by competitive binding assay and displacement of radiolabelled 125I-HO-LVA. Except one AVP tagged peptide containing a single selenium atom, all other prepared peptides were showing affinities in the similar to the ones measured for the corresponding native reference peptides. We had all required tagged peptides to proceed to the development of the analytical strategies. The design of both analysis workflow involving molecular (Partner 1) and elemental (Partner 2) mass spectrometry was performed taking into account the constraints of the pharmacology experiments. The recourse to the real biological matrix (cell cultures) allowed optimizing both pharmacology and mass spectrometry protocols in order to reach the targeted detection level. Competitive binding assays were recently carried out with membranes prepared from cell lines expressing the vasopressin receptor and tagged AVP and HO-LVA ligands (elemental and/or organic tags). These first set of experiments showed the proof of concept. The robustness of the analytical process using MALDI mass spectrometry was undertaken through statistical evaluation of the recorded data. The elemental mass spectrometry based strategy requires further optimization to achieve the targeted quantification limit.

The optimization of the detection of peptides containing selenium atoms by ICP-MS and their quantification with the use of a separation technique coupled to the mass spectrometer (LC-ICP-MS end CE-ICP-MS) is under prospect. A new equipment (LC-ICP-MS) that is supposed to improve the plasma stability under solvent conditions with high organic content and to increase the sensitivity of selenium detection, is under acquisition by Partner 2. Preliminary analyses showed an increase in the selenium response by a factor 10. Moreover, the syntheses of peptides labelled with other organic and elemental moieties will be investigated to bring alternative strategies by direct more rapid analyses not requiring hyphenated technologies. Finally, the project has been started by the study of the model receptor/ligand Vasopressin/AVP for the proof of concept. Validation of the analytical methodologies will be performed on the CCK receptor (ligand CCK4/CCKB) befor tackling the couple enzyme/inhibitor (P38 MAP-kinase/ small heterocyclic compound).

Publications to be submitted soon on both topics of research (organic and elemental mass spectrometry)

Measuring the affinity of a ligand for its target is an issue of central importance in life science research and drug development. In particular, understanding how well a potential drug interacts with its target (receptor, enzyme, DNA…) provides valuable knowledge in the search for new pharmaceuticals. Collective progress in chemistry, biology, robotic, informatics, led to the advent of high throughput screening (HTS) and enabled the biological evaluation of large number of drug candidates. As far as ligand-receptor system is concerned, binding of a drug candidate is characterized by a “competitive binding assay”. Since the affinity of reference ligand for target receptor is generally high, measurement of signal requires a very good sensitivity and as a consequence, radioactivity is still the method of choice for pharmacological studies of receptor/ligand systems. However, radioactivity implies a lot of constraints linked to radioelement manipulation and storage, strongly limiting high throughput screening applicability. In this context, we aim at developing a new and universal technology involving non-radioactive detection and quantification of the reference ligand displaced by the competing candidates. Mass spectrometry offers sensitive and selective methodologies to quantify molecules in complex biological systems. In contrast to known methods, the purpose of the submitted project is to develop a generic MS-based competitive binding assay avoiding the synthesis, for each evaluated molecule, of the corresponding quantification standard. This point is a great advantage since the isotopic standard used for quantification will be the same for all biological systems. The novelty of such an approach thus lies in the joint use of mass spectrometry and original labeling chemistries. Several robust mass spectrometry technologies (MALDI-MS, ESI-MS and ICP-MS) will be investigated in conjunction with different types of new chemical tags (preformed ions, elemental tags, MS enhancers…). The strategies will be validated with soluble proteins (for instance p38 kinases) as well as membrane proteins (such as two GPCR model systems: CCKB and MCR1), involving either small heterocyclic drugs or peptides as native ligands. If successful results are obtained during the course of the proposed basic research project, the developed generic tag/MS-based quantification protocol will have a great impact in biosciences, in particular in research laboratories dealing with pharmacology.