Liquid Chromatography-High Resolution Mass Spectrometer (LC-HRMS) for Discovery and Targeted Metabolomics/Lipidomics research
Lead Research Organisation:
The Francis Crick Institute
Department Name: Research
Abstract
The Crick is a modern medical research institute that houses 120+ research laboratories. Central to meeting The Crick's aspiration of becoming one of the world's leading research institutes is the delivery of cutting-edge science using state-of-the-art instrumentation. To achieve this, The Crick has established centralised Scientific Technology Platforms (STPs) that house this instrumentation and are supported by staff of specific expertise. This model allows operational efficiency and institute-wide consistent standards of the highest quality.
Over the last decade, metabolomics - the measurement of all small molecules (or metabolites) in a given biosystem - has emerged as a core and essential technology in fundamental cell biology research. Indeed, The Crick Metabolomics STP currently works with ~70 Crick research laboratories and for many of these, metabolomics is business critical (notably, those studying cancer metabolism, cancer progression, infection and immunity, disease pathogenesis, and developmental biology). Metabolomics can range from 'broad-stroke' discovery profiling (where as wide a range of metabolites as possible is measured) to intricate targeted analyses (e.g. metabolic pathway flux measurements). The metabolites themselves vary greatly in their chemical and physical properties, from small polar metabolites to large non-polar lipids. Metabolomics relies, therefore, on sophisticated mass spectrometry instrumentation in order to cover as much of this broad range of metabolites as possible.
To achieve this, The Metabolomics STP currently houses three liquid chromatography-mass spectrometry (LC-MS) instruments. These Thermo 'Orbitrap' instruments are well-established in the field as being the gold standard for metabolomics studies. The Orbitraps afford the speed and mass resolution power essential for this work, allowing the detection and quantification of thousands of metabolites in any given run and - importantly - better identification of 'unknown' metabolites (easily the biggest bottleneck in metabolomics today).
The STP runs ~35,000 samples per year, of which half are analysed by LC-MS. Since 2013, 36,000 have been run on our Thermo Q-Exactive Orbitrap instrument, with 15,000 in the last two years alone. This highlights not only that this is a workhorse instrument, but that its workload continues to increase.
However, this instrument is now at end-of-life and, given both the workload of the instrument and the increased risk of major failure over time, breakdown of the Q-Exactive represents a critical risk to the ability of the Metabolomics STP to function as necessary. Moreover, this system lacks the analytical capabilities of the newer generation of Orbitrap LC-MS instruments. The proposed replacement - the Thermo Vanquish-Exploris 240 - will match the capabilities of the other instrumentation currently within the Metabolomics STP. This will both allow a continuity of data quality standards and require negligible additional training for the STP staff as they are already highly experienced in this instrument.
Importantly, the Exploris 240 has considerably better specifications than the Q-Exactive. The much-improved mass resolution and scan speed will be important for the stable isotope labelling techniques in which we specialize, allowing us to differentiate between isotopes of different elements and study metabolism and metabolic pathway activity with greater depth. Moreover, the Exploris 240 has improved data acquisition capabilities, including 'Acquire X' - a mode that allows automated generation and updating of workflows for ion fragmentation ('inclusion' and 'exclusion') lists, allowing a hands-off, more in-depth feature analysis, hugely improving metabolite identification efficiency and confidence, improving capacity, and freeing up valuable time for the operator.
Over the last decade, metabolomics - the measurement of all small molecules (or metabolites) in a given biosystem - has emerged as a core and essential technology in fundamental cell biology research. Indeed, The Crick Metabolomics STP currently works with ~70 Crick research laboratories and for many of these, metabolomics is business critical (notably, those studying cancer metabolism, cancer progression, infection and immunity, disease pathogenesis, and developmental biology). Metabolomics can range from 'broad-stroke' discovery profiling (where as wide a range of metabolites as possible is measured) to intricate targeted analyses (e.g. metabolic pathway flux measurements). The metabolites themselves vary greatly in their chemical and physical properties, from small polar metabolites to large non-polar lipids. Metabolomics relies, therefore, on sophisticated mass spectrometry instrumentation in order to cover as much of this broad range of metabolites as possible.
To achieve this, The Metabolomics STP currently houses three liquid chromatography-mass spectrometry (LC-MS) instruments. These Thermo 'Orbitrap' instruments are well-established in the field as being the gold standard for metabolomics studies. The Orbitraps afford the speed and mass resolution power essential for this work, allowing the detection and quantification of thousands of metabolites in any given run and - importantly - better identification of 'unknown' metabolites (easily the biggest bottleneck in metabolomics today).
The STP runs ~35,000 samples per year, of which half are analysed by LC-MS. Since 2013, 36,000 have been run on our Thermo Q-Exactive Orbitrap instrument, with 15,000 in the last two years alone. This highlights not only that this is a workhorse instrument, but that its workload continues to increase.
However, this instrument is now at end-of-life and, given both the workload of the instrument and the increased risk of major failure over time, breakdown of the Q-Exactive represents a critical risk to the ability of the Metabolomics STP to function as necessary. Moreover, this system lacks the analytical capabilities of the newer generation of Orbitrap LC-MS instruments. The proposed replacement - the Thermo Vanquish-Exploris 240 - will match the capabilities of the other instrumentation currently within the Metabolomics STP. This will both allow a continuity of data quality standards and require negligible additional training for the STP staff as they are already highly experienced in this instrument.
Importantly, the Exploris 240 has considerably better specifications than the Q-Exactive. The much-improved mass resolution and scan speed will be important for the stable isotope labelling techniques in which we specialize, allowing us to differentiate between isotopes of different elements and study metabolism and metabolic pathway activity with greater depth. Moreover, the Exploris 240 has improved data acquisition capabilities, including 'Acquire X' - a mode that allows automated generation and updating of workflows for ion fragmentation ('inclusion' and 'exclusion') lists, allowing a hands-off, more in-depth feature analysis, hugely improving metabolite identification efficiency and confidence, improving capacity, and freeing up valuable time for the operator.
Technical Summary
The Metabolomics STP seeks to replace an end-of-life Thermo Q-Exactive liquid chromatography-mass spectrometer (LC-MS) with an instrument of improved specifications and capabilities. The instrument will be used for polar and lipid analyses, both in metabolite profiling and targeted (notably stable isotope labelling) experiments. The ability to cover a broad mass range with high resolution and scan speed is essential. Our preferred option is the Thermo Vanquish-Exploris 240. Our reasons for this are:
1. The mass resolution and scan speed of the Exploris 240 (240,000 at m/z 200 and 22 Hz, respectively) is far superior to the Q-Exactive (140,000 and 10-12 Hz) over our mass range of interest (typically 40-1250 m/z). This is critical because: i) more accurate mass measurements and, hence, identifications can be achieved - especially important in profiling experiments and the stable isotope labelling techniques in which we specialize; ii) the improved scan speed allows higher resolution acquisition and more ion fragmentation - even in polarity switching modes - and better resolution of closely eluting peaks, again allowing more accurate metabolite identification.
2. Importantly, the Exploris 240 has powerful new acquisition workflows (Acquire X), including a mode that provides automated generation and run-to-run updating of inclusion and exclusion lists with an automated re-injection ability, enabling deeper feature analysis, hugely improving metabolite identification efficiency and confidence, improving capacity, and allowing the operator to be more hands-off.
3. The Exploris 240 will either match or better the capabilities of the other LC-MS instrumentation currently within the Metabolomics STP (namely a 2-year-old identical instrument and 7-year-old Thermo Q-Exactive-plus). This will both allow a continuity of data quality standards and require negligible additional training for the STP staff as they are already highly experienced in Thermo LC-MS instrumentation.
1. The mass resolution and scan speed of the Exploris 240 (240,000 at m/z 200 and 22 Hz, respectively) is far superior to the Q-Exactive (140,000 and 10-12 Hz) over our mass range of interest (typically 40-1250 m/z). This is critical because: i) more accurate mass measurements and, hence, identifications can be achieved - especially important in profiling experiments and the stable isotope labelling techniques in which we specialize; ii) the improved scan speed allows higher resolution acquisition and more ion fragmentation - even in polarity switching modes - and better resolution of closely eluting peaks, again allowing more accurate metabolite identification.
2. Importantly, the Exploris 240 has powerful new acquisition workflows (Acquire X), including a mode that provides automated generation and run-to-run updating of inclusion and exclusion lists with an automated re-injection ability, enabling deeper feature analysis, hugely improving metabolite identification efficiency and confidence, improving capacity, and allowing the operator to be more hands-off.
3. The Exploris 240 will either match or better the capabilities of the other LC-MS instrumentation currently within the Metabolomics STP (namely a 2-year-old identical instrument and 7-year-old Thermo Q-Exactive-plus). This will both allow a continuity of data quality standards and require negligible additional training for the STP staff as they are already highly experienced in Thermo LC-MS instrumentation.
Organisations
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ORCID iD |
| James MacRae (Principal Investigator) |