The integration of instrumentation is essential to resolve cryptic challenges of analysis in clinical research and drug discovery. The advancement in information technologies, data processing and analytical instrumentation has been paving ways of resolving the issues of bioanalytical with very high precision and accuracy. The hybrid instrumentation in liquid chromatography, mass spectrometry, nuclear magnetic resolutions have heralded an era of revolution in bioanalytical field. In continuation of advancement in bioanalytical, recently Fourier Transform-Ion Cyclotron Resonance- Mass Spectrometry (FT-ICR-MS), novel hybrid instrumentation is finding versatile applications in fields like proteomics, genomic, natural product analysis, metbonomics and pharmacokinetic/ pharmacodynamic (pk/pd) studies.
Modern drug discovery and development is heavily dependent on rapid and highly efficient analytical methods. Emphasis is increasingly placed on speed and high throughput by instruments like FT-ICR-MS, however the utlization of these techniques are not upto the expectations due to mindset and over dependence of scientists on traditional methodologies. Although modern trap and time-of-trap instruments can measure many mass spectra per minute, whereas an FT-ICR-MS can do it in a second. The FT-ICR-MS is increasingly used in pharmaceutical industry due to its ability to yield more information in a comprehensive manner. This can help to analyze and search information continuously about disease mechanisms.
The high mass accuracy and resolution of FT- ICR-MS is making it an increasingly useful tool in drug discovery and development. Although FT-ICR is not a high throughput method in the traditional sense, previously difficult and complex problems are being efficiently approached using steadily improving instruments and magnets.
In FT-ICR cell there is a combination of magnetic and electric fields forces in which ions are to move in circular orbits (cyclotron motion) but however it does not restrict their motion in the axial direction along the field lines. Axial trapping plates (or rings) at a low potential (as low as e.g. 1 volt) are therefore used to keep ions with in the cell, The ion motion in an ICR-cell is a superposition of three independent motions, namely the cyclotron motion, the magnetron motion and the trapping oscillation. Only the 'reduced' cyclotron frequency is detected, by which the m/z of an ion is determined.
The magnet is the most critical component of FT-ICR -MS which determines the cost and performance. The higher the field, the better the resolution and sensitivity, so super conducting magnets are widely used. Fields of 3.0, 4.7, 7.0, 9.4 and, recently, 12 tesla are common. Modern magnets are, in general, are actively shielded.
For biologically related applications, electro spray ionization (ESI) is probably the most widely used technique. Matrix-Assisted Lasers Desorption-Ionization (MALDI) has become more popular since the high-pressure source keeps kinetic and internal energies were low. To retain resolution, ions must be trapped in the ICR-cell at low voltages, thus it is essential that kinetic energy is kept to a minimum. Low internal energy reduces metastable decay in the cell. A combined ESI-MALDI source is also now commercially available. Internal ion sources, such as Electron Impact (EI), Chemical Ionization (CI) and photo ionization, for volatile compounds, as well as Laser Desorption-Ionization (LDI) for non-volatile compounds can also be combined with FT-ICR. Atmosphere Pressure Chemical Ionization (APCI) and Atmosphere Pressure Photo Ionization (APPI) have also been applied.
FT- ICR can be directly and indirectly coupled online with different separation techniques like ESI, high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Coupling LC with MALDI-FT-ICR is usually off-line. Recently, a fully automated LC-MALDI-FT-ICR platform for proteomics enabling peptide mapping, MS-MS and complex mixture analysis in a high-throughput fashion is available. Nano-HPLC combined with Nano-ESI-FT-MS has been implemented for characterizing complex protein mixtures without pre-fractionation.
For high mass accuracy, internal mass calibration is the typical approach. Some instruments provide internal calibration by mixing calibrant and analyte ions in the ESI emitter, which causes ion suppression and reduces the effective dynamic range. A new approach with independent ionization, introduction and transmission control of analyte and calibrant species has shown much better performance for MALDI and ESI sources.
The triple quadrupole-FT-ICR hybrid enables data dependent ion pre selection, external fragmentation (MSn) data-dependent MSn, and can be online coupled with HPLC. Combining linear ion traps triple quadrupoles with ICR makes it possible to perform automated ultra-high resolution, high mass accuracy and MSn in biological applications.
Applications of FT-ICR-MS
Profiling of proteins (proteomics) has led to a host of biomedical applications relevant to drug discovery, particularly for target identification and diagnostic profiling. Mass spectroscopy is an indispensable tool for proteomics and FT-ICR is gaining in importance because of the unique possibilities it presents. Unmatched mass accuracy (~1 ppm versus ~10 ppm) and resolution (>106 versus ~104) lead to higher confidence in protein identification. In FT-ICR-MS Measurement, a fewer number of peptides required for protein identification. The De novo sequencing is possible with only few fragmentsions. With this technique facilitation of protein structure experiments is possible. Thermal and non-thermal MSn enables top-down proteomics. Since only protein backbone is cleaved so that the Post Transnational Modifications (PTMs) remain intact which helps in location of PTMs.
FT-ICR has proven to be a powerful tool in the study of noncovalent complexes, particularly because the method is well suited for high-throughput affinity screening, and various types of complexes have been analyzed. For example, RNA has been used as a target for drug discovery in an assay called multitargeted affinity-specificity screening.
FT-ICR has become versatile tool for natural products based drug discovery programme. Natural products are a rich source of medicinal compounds. Nearly half the major drugs on the market are derived from natural products. FT-ICR is heavily used for structure elucidation or verification, particularly for antibiotics. FT-ICR with electron impact ionization has been used to establish the structure of a tumour inhibitor isolated from dried Euphorbia pubescens . Lead identification and optimization is the most challenging fields in drug discovery. The large peak capacity of FT-ICR is an advantage for the characterization of combinatorial libraries. FT-ICR has been used to characterize mucin-2 antigen peptide libraries, or to study target antigens related to AD for the design of vaccine peptides. FT-ICR was also coupled to capillary liquid chromatography to enable the screening of libraries with more than 1000 different peptides in less than 20 min. The screening of substrate libraries by enzymes using combinatorial libraries in combination with FT-ICR has been reported. FT-ICR was also utilized to identify the coding tags attached to a single-bead used beads from peptide-encoded combinatorial libraries with MALDI-FT-ICR in HTS.
New applications are likely to be developed by combination of FT-ICR with other techniques, or by development of ingenious methods to observe systems previously accessible. Developing applications of FT-ICR in the areas of mass spectrometry imaging, elucidations of macromolecular 3D conformations, molecular recognition spectroscopy of trapped ions and combination with prefilter stages, such as traps are some of these hot topics. Macromolecular complexes, molecular conformations and intermolecular interactions all kinds are currently developing particularly rapidly. Sensitivity and resolution will continue to be pushed to new extremes, via new ion sources and interfaces, and improved ICR cells, magnets and electronics.
All technologies continually advance, and time-of-flight (TOF) and trap instruments, in particular, have improved dramatically in recent years. Nevertheless, in some performance categories, and for a range of analysis techniques, FT-ICR will probably retain its lead for the moment. In addition, a trend toward less complex control software will reduce the barrier to routine use.
The questions confronting drug discovery are rapidly becoming more complex because the 'low hanging fruit' well-understood, readily treatable diseases are believed have been largely 'picked'. Therefore, it seems probable that FT-ICR will become more valuable because of its ability to assist in the solution of complex questions. From protein activity modulation via PTMs to intermolecular complexes and binding sites to high-order structures to biomarker discovery and pathway recognition, FT-ICR has capabilities that are not readily matched in other instruments. u
-- The author is with Pharmacy Group, Birla Institute of Technology and Science, Pilani, Rajasthan