The next generation
of hybrid linear ion trap mass spectrometers

The development of mass spectrometers has advanced to such a degree over the last ten years that it is hard to think of an application where they are not used. Within the life science industry, in particular, they have become a ubiquitous tool for proteomics and small molecule applications.

For all of these advances in technology, a majority of mass spectrometers cannot obtain all the information that is required and two or more techniques must often be combined to achieve maximum sensitivity and throughput. Take, for example, quadrupole and ion trap analysers. For some applications it is difficult to obtain good quality spectra with a triple quadrupole instrument, as the sensitivity in product ion mode is often not sufficient. This lack of sensitivity can be overcome with QqTOF technology, which gives additional mass information, but true precursor and neutral loss scan experiments and reliable quantitative analysis is not possible. The same is true of 3D ion trap technology, although ion trap mass spectrometry (MS) does allow sensitive multiple MS (MSn) experiments to clarify the fragmentation process, simplifying spectral interpretation.

However, 3D ion traps can easily become overloaded with ions (space charge effects), which can lead to mass shifting, resolution loss and non-linearity in the spectra and calibration curves. As a result, 3D ion traps are less suitable for quantitative analysis This all changed with the introduction of the first hybrid linear ion trap/triple quadrupole mass spectrometer, the Q TRAP LC/MS/MS System from Applied Biosystems/MDS SCIEX. The Q TRAP System, which is based on the API 2000 LC/MS/MS platform, has been rapidly accepted, in particular by the proteomics and drug discovery and development mass spectrometry markets. It has a big advantage over the traditional 3D ion trap instruments in that the technology is much less sensitive to space charge effects as the ion storage capacity of the linear trap is approximately 45 times higher. Moreover, there is no low mass cut-off for the Enhanced Product Ion (EPI) scan and sensitivities that have been traditionally achieved with Multiple Reaction Monitoring (MRM) can now be achieved using EPI scans. Having high- sensitivity quantitative and qualitative performance on one instrument opens up the possibilities to a) combine experiments that previously had to be run on two different MS technology platforms and b) to obtain data that was previously not easy to acquire. The introduction of the 4000 Q TRAP LC/MS/MS System took the technology a step further, offering all the same capabilities of its predecessor but between 10 to 50 times more sensitive, depending on the application. It provides rapid, automated quantitative and qualitative analyses and, as such, has applications in the pharmaceutical, food, environmental, clinical research, forensic, toxicology and metabolic profiling industries. The system also has a higher mass range (up to m/z 2800) making it uniquely suited for the automated analyses of post-translational modifications (PTMs), as well as protein identification and peptide quantitation. There are four different ion sources available with the 4000 Q TRAP System; the TurboV source, a Duo-Spray source, which is a combination of TurboIonSpray and APCI in a single source to allow rapid (i.e. 100 ms) switching between the two ionisation technologies during the liquid chromatography (LC) run, the Photospray source for Atmospheric Pressure Photo Ionization and a Nanospray source for off-line and on-line peptide and protein analysis. There are also several add-on software packages available, such as Metabolite ID for automated identification and characterisation of metabolites, BioAnalyst software and ProID software for proteomic applications.
MS in forensic toxicology
An example of the power of the instrument can be demonstrated by its application to routine screening in forensic toxicology, where the objectives are to:
-screen and quantitate common and prohibited drugs from blood, urine and saliva samples
-obtain structurally significant information on the drug compounds in the same chromatographic timescale for confirmation purposes
-analyse patient-related extracts and provide positive identification of any drugs that are detected.
In the past, this type of analysis would have typically been run on a triple quadrupole in MRM mode for selective and quantitative target compound analysis. In addition, to obtain structural information, a 3D ion trap would be required because of its full scan sensitivity and MS3 capability. With the 4000 Q TRAPSystem, however, all information can be obtained with one system and in a single run. This is accomplished using the Information Dependant Acquisition (IDA) software package which has been implemented in the Analyst 1.4 acquisition software. IDA combines the information from survey scans into the decision-making process to take advantage of the specificity of scans such as precursor ion and neutral loss while maximising data collection. Figure 1 shows IDA looped multiple level experiments for the entire duration of the LC analysis. Among the criteria available for the IDA ion selection, specific mass and retention time (SMART) filters can be applied for the inclusion or exclusion of ions of interest. This provides maximum flexibility to the user when isobaric interferences are found or for rapid specific analysis when MS/MS confirmation is required. Enhanced resolution could be added as a confirmation scan to obtain MS information with higher resolution. This information is particularly useful for charge state (z=1 to 5) determination and isotopic ratio measurements when used after scans (precursor or neutral loss), which may not reveal this information. Since the 4000 Q TRAP System has the ability to collect MS3 data, a second level of dependency was implemented in the loop. Thanks to the LINAC collision cell the system is also able to handle multiple MRM transitions, which allows a large number of compounds to be screened simultaneously without loss of sensitivity or cross-talk phenomena. In the following example (see figure 2 & 3), a spiked urine sample was screened for nine common tranquillisers. A five-minute liquid chromatography (LC) gradient at 500 µl/min was run and the nine tranquillisers were screened using one MRM transition for each tranquilliser. When a positive signal was detected for a specified MRM transition, the software automatically acquired the enhanced product ion MS/MS spectrum for each tranquilliser thereby providing structural information to confirm the identification of the compound. By combining experiments on one system and using automated software tools like IDA to perform analyses in one LC run, sample analysis time is reduced and there is no need to reproduce LC conditions on different MS platforms.
Similarly, being able to automatically combine sensitive triple quadrupole and ion trap scanning in a single analysis has advantages when it comes to the identification and sequencing of post-translational modifications (PTM), in particular phosphorylation. For phosphorylation analysis, a negative ion precursor ion scan for m/z 79 identifies the unique phosphoric acid loss from phosphopeptides. This precursor ion scan is used as a survey scan during automated IDA. Ions present in this precursor scan are then analysed automatically in the positive ion with an enhanced resolution scan, followed by a high sensitivity MS/MS scan to identify the phosphopeptide and the site of phosphorylation. Pro ID software is used to determine the protein of origin for each modified peptide and to identify the site(s) of modification on the peptides.
Conclusion
The 4000 Q TRAP system provides extremely sensitive quantitative and qualitative capabilities in one instrument.
It offers unmatched qualitative performance as well as the sensitivity and wide dynamic range required for quantitation. The intuitive application specific software is a powerful easy-to-use tool that allows 24 hour, high throughput operation and, with a full complement of automation features, it can also boost productivity.