Gary Impey

LC–MS-MS fractionation experiments or direct electrospray infusion can generate information-dependent MS-MS data triggered from a suite of triple-quadrupole selective scans or linear ion trap (LIT) scans. As shown in Figure 1, precursor ion (PIS), neutral loss (NL), multiple reaction monitoring (MRM), or enhanced MS LIT scanning can trigger an information-dependent enhanced product ion scan for MS-MS characterization of lipid structures.  

Faster quadrupole scanning and LIT scanning (up to 20,000 Da/s) combined with faster polarity switching and sensitivity improvements in enhanced product ion and MS

  scanning in LIT mode provide qualitative and quantitative strategies simultaneously. Figure 2 shows the coupling of scans for global lipid profiling on the AB SCIEX QTRAP 5500 system. Methods consisting of a suite of survey scans triggering multiple MS-MS scans yield in-depth lipid coverage in samples of wide dynamic range. 

Targeted Multiple Precursor Ion Scanning by Direct Electrospray Infusion

A total of 60 combinations of PIS and NL multiplexed experiments can be acquired simultaneously for comprehensive quantitative analysis of lipid species by direct infusion. An example of data collected with negative mode multiple ion method consisting of multiplexed scanning of fatty acid acyl ions of varying saturation and carbon chain length, and diagnostic fragments for selected lipid classes, is shown in Figure 3. These data can be used directly to determine the abundance of lipids in a particular class from class-specific precursor ion experiments or to design subsequent MRM methods to quantify a subset of lipids of interest. 

Measuring the Quantitative Response of Lipids to Class-Specific Internal Standards

Lipid class internal standards are essential to confirm, normalize, and quantitate corresponding lipid species that were detected with the MS approaches mentioned earlier (1). To study the linear dynamic range, a series of samples (three replicates for each concentration) consisting of the internal lipid class standards (held constant at 0.15 μM) and an increasing amount of total lipid extract from 10

  μg were prepared from brain tissue. LIT enabled enhanced MS information-dependent acquisitions (IDA) triggered MS-MS methods were carried out and data were processed by LipidView software to evaluate the accuracy and precision of the quantitation. Figure 4 illustrates a linear response of representative lipids once corrected for their corresponding lipid-class specific internal standards across the calibration line over five orders of magnitude. Specifically for phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) lipids, the limits of detection are well in sub pmol level. 

Post-Acquisition Data Processing for Lipid Identification and Quantitation

LipidView software is data processing software that identifies and quantifies lipid species from ESI-MS data. The software enables identification and characterization of lipid species through searching masses of parent and fragment ions in a database of characteristic fragments and neutral losses corresponding to parts of lipid molecules, and reports a numerical and graphical output for profiling various lipid molecular species, lipid classes, fatty acids, and long-chain bases (3). The software streamlines various steps, such as automated data processing method selection, lipid species identification, comprehensive isotope contribution removal, multiple internal standards-based quantification, visualization, and reporting of the results. A view of the user interface is shown in Figure 5 demonstrating the results workspace for reviewing the lipid profile data from a processed raw data file. 

Improvements in Speed and Sensitivity for Lipidomics Applications

The AB Sciex QTRAP 5500 System's speed, selectivity, and sensitivity enable high multiplexing of QqQ scanning, and whole lipid extracts can be analyzed by either direct infusion of LC methods for in-depth lipid profiling — achieving both qualitative and quantitative data (with the use of synthetic lipid internal standards) in very short acquisition times. In addition to sensitivity gain, the MS system provides a polarity switching time of 50 ms, a linear accelerator trap scanning rate of 20,000 Da/s, and an LIT fill time of 50 μs. Lastly, Analyst 1.5 Software using the Scheduled MRM algorithm enables up to 2500 MRM transitions monitored in a single experiment for lipid quantitation in LC-based workflows. 

Scientists require versatility, sensitivity, specificity, and speed for high-throughput lipidome profiling of complex biological extracts by either direct infusion electrospray or LC–MS methods. For optimal results, data acquisition strategies should be coupled with automated data processing for lipid species identification and quantitation. 

 ar are with Applied Biosystems|MDS Analytical Technologies, Toronto, Ontario, Canada. 

(1)  M. Stahlman, C.S. Ejsing, K. Tarasov, J. Perman, J. Boren, and K. Ekroos, 

(2)  D. Schwudke, J. Oegema, L Burton, E. Entchev, J.T. Hannich, C.S. Ejsing, T. Kurzchalia, and A. Shevchenko, 

(3)  C.S. Ejsing, E. Duchoslav, J. Sampaio, K. Simons, R. Bonner, C. Thiele, K. Ekroos, and A. Shevchenko, 

Articles

The authors discuss analytical methods for lipidomics.

Accelerated Discovery and Quantitation of Lipids in Complex Extracts

Laboratories embroiled in the intricacies of drug discovery and development, proteomics, and food and water testing are becoming increasingly dependent upon sophisticated mass spectrometers to overcome the significant and limiting challenges they face. Problems such as sample type, quality, and concentration, ever-increasing throughput demands, and limitations of currently available mass spectrometry workflows are all a matter of course and must be dealt with to push the boundaries of analysis even further.

For drug discovery workflows, the issue of metabolite detection and identification in in vivo systems is a critical challenge. The wide range of complex matrices (such as bile, plasma, urine, and fecal extracts), and the ion suppression effects of these biological fluids, can cause a severe decrease in the ability to detect metabolites. Greater instrument sensitivity is necessary to detect these compounds and, at the same time, helps to minimize sample preparation, simply diluting the negative effects of these complex matrices and avoiding the time- and labor-consuming sample cleanup or concentration steps that otherwise might be required. 

In drug development, microdosing (using less than one hundredth of the normal dose) studies allow earlier, and potentially safer, assessment of the pharmacokinetics of new compounds in humans compared to conventional Phase I trials, as well as to reduce or replace safety and toxicology testing in animals. The high sensitivity of liquid chromatography–mass spectrometry (LC–MS) is a significant factor in such strategies, allowing the detection of much lower concentrations of compounds and metabolites in both quantitative and qualitative work. 

Many of the compounds of interest occur in low levels, a familiar issue in biomarker verification and validation work too, and insufficient instrument sensitivity could lead to the failure to detect key compounds. In the food and environmental industries also, quality control processes constantly demand greater sensitivity to keep apace with legislative regulations that progressively reduce acceptable limits of an increasing number of contaminants. With the large number of compounds that must be detected, less time is available to monitor each compound within a run, resulting in poor sensitivity and often leading to multiple injections, adding considerable time to experiments. Improved instrument sensitivity and scan speed would allow many more analytes to be monitored in the same run at the required sensitivity so that fewer injections would be required per experiment, thereby offering the potential to obtain all the necessary answers in a single injection. The speed of the mass spectrometer is also important in other ways. With the current trend for faster chromatographic analysis times of 5 min or less, a system must be capable of yielding sufficient data points from each chromatographic peak for effective LC–MS analysis. In addition, faster scanning and improved duty cycle enable more efficient automated workflows. 

For the identification of a large number of compounds (for example, in the pharmaceutical discovery phase), a first pass traditionally has been performed with a triple-quadrupole mass spectrometer for the selective scan functions, like precursor ions and neutral loss, helping to filter out unwanted compounds. In general, the selective nature of multiple reaction monitoring (MRM), precursor ion, and neutral loss scans provides the highest level of detection compared to any single MS-based methodology. Then, for confirmation, other instruments like ion traps were used to obtain high quality MS-MS spectra. 

Now, with a drive for greater quality, efficiency, throughput, and confidence in data using as few injections as possible, mass spectrometers are being designed to meet scientists' needs precisely. Instruments with improved sensitivity, speed, and selectivity, coupled with unique combinations of technologies within a single platform to enable more efficient workflows, are being developed by leading manufacturers. Hybrid instruments include Applied Biosystems/MDS Analytical Technologies' AB SCIEX QTRAP 5500 System, which combine a triple quadrupole and a linear accelerator trap on the same platform. Such instruments are changing the way MS analysis, like metabolite identification, is performed (for example, predictive MRM [pMRM], coupled with full-scan MS-MS in the same analysis to confirm that the compound is the expected metabolite). The hybrid nature also allows more than one test to be performed in one run, which is ideal for applications such as testing for pesticides and mycotoxins in grain-derived food products, and screening for pharmaceuticals and personal care products (PPCP) in drinking water. 

With the addition of a newly developed ion guide and improved Q3 LIT, the latest of these systems shows great increases in signal response, particularly in LIT mode. Duty cycle is improved further with trap fill times as short at 50 μs, because of the increase in linear accelerator trap sensitivity. We are also seeing scan rates up to 20,000 Da/s in linear accelerator trap mode and four times faster in quadrupole mode compared to previous systems. This greater cycle rate allows for compatibility with sharp chromatographic peaks, as well as improved information-dependent acquisition (IDA) coverage provided by a greater number of MS-MS-dependent scans, a great advantage when complex matrices, such as bile, contain a notable number of nonmetabolite peaks and may cause extraneous IDA triggers. The improvements in triple-quadrupole scanning rates also allow multiple survey scans, and simultaneously trigger full-scan MS-MS from any peaks that might be present using LIT. The greater speed of the newest instruments, which can detect more than 1000 compounds, means that they are ideal for multitarget analysis, detecting all compounds of interest in just one injection (Figure 1). 

 Figure 1: Analysis of grape samples for more than 1000 pesticides. Methomyl and trifloxystrobin were quantified and identified using scheduled MRM and fast MS-MS library searching. 

With polarity switching as fast as 50 ms, it is possible to combine the most powerful modes of metabolite detection in a single injection workflow with sufficient sensitivity in an overall duty cycle time of around 2 s, for an accurate result in comprehensive detection of metabolites (Figure 2) and pesticides (Figure 3). 

Figure 2: Detection of simvastatin hydroxyl acid in both positive and negative ion. The lactone prodrug simvastatin is hydrolyzed to the active compound (simvastatin hydroxyl acid), and both simvastatin and the hydroxyl acid ionize in positive ion, but the hydroxyl acid is better detected in negative ion. Of the 34 metabolites, five were detected only in positive ion, 28 detected only in negative ion, and one (Mx) was detected in both polarities. 

The improved sensitivity and speed are also important factors for efficient targeted peptide quantitation workflows, including biomarker verification and validation, and the high selectivity for compounds of interest certainly increases the throughput of automated workflows. The newer combined systems enable hypothesis-driven verifications, removing the need for synthetic protein standards in assay development, and the heightened sensitivity enables the linear accelerator trap to match the sensitivity of the MRM, speeding up development and identification confirmation. Scheduled MRM enables much higher multiplexed assays while still maintaining analytical reproducibility; with the latest instruments capable of up to 2500 MRMs in a single run, equating to 100 proteins, a single run is enough to obtain all the data required, eliminating what was previously a potential bottleneck. 

Figure 3: Ultrafast LCâMS-MS analysis of four pesticides in drinking water at 0.1 mg/L using fast polarity switching and fast MRM detection. 

The latest software programs that accompany MS platforms have been developed to bring all the sophisticated, complex methods right to the researcher's fingertips, with just a few clicks of the mouse. User-friendliness is a key feature, helping to make MS technology accessible to many more users. Ready-to-use, preconfigured protocols and experiments are especially helpful to those who are new to LC–MS, and allow them to be running meaningful experiments within weeks, rather than months.  

Data analysis software similarly has been upgraded to handle the huge amounts of data resulting from the greater throughput of new MS instruments, streamlining and automating the analysis process to require minimal manual intervention.  

The new systems offer a complete solution for LC–MS-MS workflows in various applications, with the best of both quantitative and qualitative accuracy and reproducibility, as well as generating superior targeted quantitative analysis, validation, and confirmation in a single run. 

 are with Applied Biosystems, Inc., Foster City, California. 

The authors discuss the challenges presented by the many new applications of mass spectrometry.

Gary Impey

Articles

Accelerated Discovery and Quantitation of Lipids in Complex Extracts

A New Perspective on the Challenges of Mass Spectrometry