Broadening your small molecule horizons

The capabilities offered by new generations of high performance TOF and TOF/TOF mass spectrometers open up a breadth of new research and applied market opportunities for the analysis of small molecules says Michael Willett 

EUROPEAN research scientists’ use of advanced Electron Transfer Dissociation (ETD) on high-capacity ion trap mass spectrometers is becoming widespread, with a large number of high-performance ETD user installations now in universities, pharma and biotech. With the recent availability of new more advanced ion trap software, high-capacity ion trap Post Translational Modification (PTM) discovery research can now be performed on ion trap mass spectrometers (MS) fully equipped with both ETD and PTR (Proton Transfer Reaction) capabilities. With exciting new unique and innovative ion optics designs and chemistry setups for ETD/PTR, rapid, routine top-down characterisation of large peptides and mid-size proteins in high-capacity ion traps has become routine for the research scientist.

The new generation of advanced ETD on high-capacity ion trap mass spectrometers is revolutionising proteomics mass spectrometry, as demonstrated by the very significant success of these systems in the proteomics market. The simplicity and vastly improved sensitivity of doing advanced ETD in a high-capacity trap, which traps positive and negative ions at the center of the ion trap configuration, makes for ETD implementations which are robust, highly sensitive, fast,  and most useful. It is difficult to imagine any future cutting-edge European proteomics laboratory without these dramatic new capabilities for de novo sequencing, greater sequence coverage, and most importantly, sensitive, and fully automated PTM discovery and localisation. ETD on a high-capacity trap is a nearly ideal combination for modern proteomics research.

The usefulness of ETD/PTR has been well demonstrated by the unambiguous characterisation of PTMs. New ion trap software has introduced a novel Collision Induced Dissociation (CID) fragmentation mode which eliminates the well-known low mass cut-off of ion traps in MS/MS. It enables multiplexed quantitative proteome analyses by leading labeling chemistries on high-capacity ion trap systems. Moreover, a new AutoMSn mode in the new ion trap software automatically eliminates the most abundant ions in order to further enhance dynamic range.

Finally, scheduled target lists in new ion trap software extend the number of possible Multiple Reaction Monitoring (MRM) transitions per run. This new dynamic scheduling allows monitoring hundreds of target compounds in parallel for large scale biomarker validation studies or for pesticide, drug and doping screening.

With a new generation of proteomics data-warehousing software now in use, next-generation bioinformatics platforms addressing scientists' current needs in biomarker profiling, quantification and validation have become available. A comprehensive solution for qualitative and quantitative liquid chromatography-MS/MS protein analysis can now support all current label chemistries including multiplexed labels, as well as label-free quantification. Interactive validation of protein quantification based on raw LC/MS data has become simple and straight forward. It streamlines the discovery process through decoy auto-validation algorithms and algorithms that produce non-redundant protein result lists across entire proteomics projects.

New proteomics data-warehousing and project management software even has a number of dedicated data viewers that permit the evaluation and validation on each level of proteomics experiments, such as LC/MS survey viewers, the gel viewers and sequence-annotated MS/MS spectra. All these views are linked and permit simple browsing through scientists' proteomics data, supported by extensive queries. Software can now allows the retrieval of data generated years ago, allowing their joint re-analysis with novel analytic capabilities and mining tools.

Any new proteomics data-warehousing software, when used with brain proteomics research, is expected by researchers to be fully compliant with the HUPO Brain Proteomics Project processing guidelines (forum.hbpp.org), eventually facilitating the direct submission process of project data adhering to HUPO/PSI publishing guidelines.

New MALDI molecular imaging systems are now used for biomarker detection, tissue classification and pathology research, as well as for imaging the distributions of drugs and metabolites in drug development. The novel statistical analysis of tissue images enables the concept of MALDI class imaging, the molecular imaging equivalent of diagnostic multimarker panels. Recent data on biomarker discovery, obtained with MALDI molecular imaging mass spectrometry systems, demonstrate the power of direct tissue analysis by MALDI-TOF (Time of Flight) and MALDI-TOF/TOF high-end mass spectrometers.

State of the art MALDI molecular imaging solutions now include new advanced image preparation devices for automated high-resolution matrix application on tissue, smart laser beam technology and sophisticated class imaging algorithms for tissue classification and biomarker detection and molecular histology, as well as imaging of drug and metabolite distributions in drug development.
Mass spectrometry firms are commiting to developing innovative, far reaching proteomic applications. They are focusing on some of the most important areas to proteomics scientists, like high-sensitivity PTM discovery, as well as biomarker discovery, quantification and rigorous validation.

New MALDI molecular imaging sample preparation technology is now available for automated matrix deposition onto tissue slices. MALDI mass imaging of tissue samples now allows color-coded visualisation of the distribution of peptide or small protein biomarkers, or of drugs and their major metabolites for the direct analysis of molecular distributions in tissue sections. The new image preparation technology provides highly reproducible sample preparations for MALDI imaging in a fully automated, push-button process. Another unique advantage is the combination of excellent spectra quality at high image resolution of 50μm at the same time, for example.

With the availability of this new imaging sample preparation technology, research scientists can now use complete MALDI molecular imaging solutions consisting of high-performance MALDI-TOF(/TOF) mass spectrometers with smart laser beam technology and enhanced panoramic mass resolution over a broad mass range, plus sophisticated software for data analysis, visualisation and statistical analysis of MALDI images, including advanced class imaging  methodology for the molecular imaging equivalent of diagnostic multimarker panels.
With the novel image preparation technology of vibrational vaporisation, the lack of robustness and the limited spatial resolution of older matrix deposition technologies has finally been overcome. Optical sensors are used to monitor light scattered by the matrix layer to greatly enhance reproducibility and quality control in MALDI tissue preparation. This quality assurance process leads to reliable and reproducible push-button sample preparation for high-information content, high resolution MALDI imaging.  Such image preparation device capability is now an essential tool for scientists and clinicians using MALDI imaging as a routine research technology, emphasising the industry’s expertise and commitment to develop advanced clinical research solutions based on mass spectrometry.

Nanotechnology-enabled matrix-free target plates for use primarily with laser desorption ionisation (LDI) time-of-flight (TOF) mass spectrometers have now become available. These plates are known as Capture and Analyse (CA) chips.
These CA chips enable significant sensitivity, throughput and ease-of-use benefits for the analysis of small molecules such as drug compounds, small peptides, natural products, pesticides or many other low mass molecules that previously were difficult to analyse with the ease and throughput of MALDI-TOF mass spectrometers. Until just recently, these molecules typically have had to be analysed with LC-MS, which is more time-consuming, somewhat less robust and requires more operator training.
CA chips, combined with robust, very fast, and exceptionally easy-to use TOF or TOF/TOF mass spectrometers, have the potential to revolutionise small molecule mass spectrometry in many important areas of life science or chemical research and analysis. Potential applications areas include chemistry and molecular biology research, molecular diagnostics and molecular imaging research, pharma/biotech and other industrial research and analysis, as well as many applied fields, such as food analysis, environmental analysis, and toxicology.
The unique capabilities offered by such CA chips, in combination with new generations of high performance TOF and TOF/TOF mass spectrometers, open up a breadth of new research and applied market opportunities for the easy, fast and robust analysis of small molecules. 

The new generation of Fourier Transform Mass Spectrometers (FTMS) now available sets a standard for high-performance FTMS in top-down proteomics and complex mixture analysis.  These FTMS superconducting mass spectrometers are now capable of sub-ppm mass accuracy and resolving powers of greater than 900,000 at 7 tesla magnetic fields, or greater than 1,500,000 at 9.4 tesla, for interrogating proteins and protein fragments, as well as complex mixtures of small molecules in metabolomics or petroleomics. Even higher resolving power and better mass accuracy are available at 12 tesla or 15 tesla. The FTMSs can now run under a unified software environment for all life science mass spectrometers with an intuitive GUI and outstanding bioinformatics capabilities. FTMS mass spectrometers are widely used in pharmaceutical research.  Developments are already well underway to make ultra-high resolution molecular imaging available on FTMS mass spectrometers, an ideal solution for small molecule molecular imaging, while MALDI-TOF molecular imaging is ideal for large molecules. 

By Michael Willett. Michael is public relations officer for Bruker Daltonics