Part 3: Solving the top 5 pain points of translational metabolomics researchers
This is the third in a multi-part blog series highlighting the key attributes and impressive benefits of the Stellar mass spectrometer—the first-of-its-kind instrument designed and optimized for highly multiplexed targeted quantitation, ideally suited for discovery verification. By delivering robust analytical methods and dramatically improved turnaround time, this solution helps labs boost capacity and differentiate themselves to generate more revenue. For additional background, see blog Part 1 and Part 2 here and stay tuned for more blogs in the series
Metabolomics is a powerful way to gain insights into a person’s biochemistry, microbiome, exposome and lifestyle. However, developing clinically relevant metabolomics panels using liquid chromatography coupled with tandem mass spectrometry (LC-MSn) presents significant challenges. That’s why clinical research labs and CROs continue to explore more effective solutions to expedite method creation and refinement, which can deliver targeted quantitative metabolites methods at scale that extend the dynamic range while using faster chromatographic methods.
By investing in new technology, your lab can deliver faster turnaround times—boosting lab capacity and ROI—while differentiating your services to attract more customers.
How new technology solves 5 key challenges for metabolomics researchers
Compared to existing triple quadrupole mass spectrometers (QQQ MS), the Thermo Scientific Stellar mass spectrometer (MS) is the most advanced solution available today for translational metabolomics research. One significant difference between the two platforms is the Stellar mass spectrometer’s ability to acquire parallel reaction monitoring (PRM*) spectra vs selected reaction monitoring (SRM*), also known for the marketing term multiple reaction monitoring (MRM*) data.
Examples of these attributes are presented in the following resources:
- Development and implementation of a comprehensive fecal metabolites LC-MS library for dietary intervention studies using the Thermo Scientific Stellar mass spectrometer.
- High throughput targeted metabolomics library generation on a novel mass spectrometer applied to microbiome analysis.
Let’s take a look at five challenges the Stellar MS solves…
Challenge #1: Overcoming tremendous chemical/structural metabolite diversity to create an effective quantitative method for the specified metabolite panel.
Mass spectrometers must perform equally well in both positive and negative ESI polarities to maximize overall quantitative performance.
- Current state: Unfortunately, some mass spectrometers can struggle with sensitive detection in ESI mode or require two different experiments (e.g., APCI and ESI) to be performed with different solvent systems, resulting in more experiment time and money.
- A better solution: The Stellar MS has an efficient ionization source and detector system to perform equally well in ionization, ion transfer and detection for both positive and negative polarity, enabling more analytes to be quantified using faster gradients.
Mass spectrometers must also have fast polarity switching capabilities to maximize the duty cycle.
- Current state: Triple quadrupole mass spectrometers may achieve 5 ms polarity switching but also require up to 5 ms for settling times before data acquisition in the second ionization polarity. That equates to 20 ms of cycle time that cannot be devoted to metabolite/lipid data acquisition.
- A better solution: The Stellar MS delivers true 5 ms polarity switching (5 ms for both polarity switching and settling times), requiring much less cycle time that can then be devoted to increasing the sensitivity of measuring targeted analytes.
Analyte characterization requires MS-level analysis to determine in-source fragmentation, adduct formation (e.g. sodium, potassium or ammonia) or if there’s an abundant co-eluting isobar or analyte. This becomes critical to identify the optimal precursor m/z value used for tandem MS or determine if LC gradient modifications are required to meet the experimental scope.
- Current state: Triple quadrupole mass spectrometers acquire full scan data slowly and with compromised sensitivity, reducing the effectiveness, even when limiting the precursor mass range transmitted through Q1 to expand beyond protonation/deprotonation. This often reduces LC-SRM (or MRM) strategies to be used for troubleshooting which is inefficient and often ineffective.
- A better solution: The Stellar mass spectrometer can acquire fast and sensitive full scan MS spectra in either polarity enabling greater analyte characterization as well as detection of abundant co-eluting matrix interferent.
Structural diversity requires greater instrument flexibility to generate diagnostic product ions associated with the target analyte structure. HCD* is the fastest fragmentation method and suitable for over 80% of targeted analytes; yet some require too high of a voltage drop, resulting in greater secondary and tertiary fragmentation, and ultimately, increased scatting that reduces product ion transmission, sacrificing sensitivity.
- Current state: Existing triple quadrupole mass spectrometers are limited to HCD fragmentation. Triple quadrupole mass spectrometers that have Qtrap functionality can perform collision-induced dissociation (CID*) but with compromised sensitivity and exceptionally longer acquisition times reducing its utility in higher multiplexed quantitation methods.
- A better solution: The Stellar mass spectrometers’ unique instrument design expands dissociation methods incorporating both beam-type higher energy collision dissociation (HCD*) as well as resonant RF CID. (Schwartz, J. C. et al. A two-dimensional quadrupole ion trap mass spectrometer 2002; 13: 659-669).
Challenge #2: Achieving enhanced specificity in the presence of complex biological matrices to maximize data confidence.
- Current state: To solve this challenge today, some researchers use alternative fragmentation mechanisms. However, existing triple quadrupole mass spectrometers are limited to HCD fragmentation, which may be insufficient for some steroids and bile acids. Additionally, triple quadrupole mass spectrometers that have Q-trap functionality can perform CID but with compromised sensitivity and exceptionally longer acquisition times (ca. 3 Hz published acquisition rates) reducing its utility for quantitative methods targeted increased numbers of analytes.
- A better solution: The Stellar mass spectrometers’ unique instrument design expands dissociation methods incorporating both HCD as well as CID. (Olsen, J. V. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed Mol. Cell. Proteomics 2009; 8: 2759-2769).
Another issue is that some analytes may require higher MSn levels to overcome background interference.
- Current state: Unfortunately, standard triple quadrupole mass spectrometers are limited to SRM/MRM*, which is MS2 acquisition. And triple quadrupole mass spectrometers that have Qtrap functionality can perform CID-based MS3 or MRM3 but with compromised performance as described above.
References: MRM3 quantitation for highest selectivity in complex matrices or Powerful scan modes of QTRAP system technology - A better solution: The Stellar MS can perform fast and sensitive MS2 (at rates up to 140 Hz) and MS3 (at rates up to 40 Hz) as well as higher-order MSn (up to MS10 if needed) using either HCD or CID at any MSn level.
Challenge #3: Current translational metabolomics experiments seek to expand sample coverage beyond 500 metabolites and/or lipids in a single method while maintaining greater sample throughput.
SRM/MRM acquisition strategies must acquire at least two or three sequential transitions for data confidence, thus doubling or tripling the number of scan events acquired per unit time. In addition, each SRM/MRM scan event has a switching time in addition to the dwell time that collectively reduces the duty cycle per analyte.
- Current state: Unfortunately, QQQ MS that can perform trapping are slow and have reduced sensitivity relative to its MRM acquisition methods.
- A better solution: The Stellar MS acquires PRM* for MSn scan events. Therefore, one scan event is needed per analyte, which increases the duty cycle and ensures qualitative analysis for high data confidence. Coupled with synchronized, dual ion packet management, Stellar MS boosts PRM acquisition speed up to 140 Hz with fast ion, sensitive detection to almost 90% of trapped ions.
Challenge #4: Maintaining the quantitative performance at scale while addressing throughput requirements during scheduled retention time (RT) windows.
- Current state: Triple quadrupole mass spectrometers do not have robust dynamic RT adjustment capabilities—requiring longer RT windows and increasing the number of concurrent SRM/MRM transitions per RT window that ultimately limit LOQ performance.
- A better solution: The Stellar mass spectrometer has the unique ability to perform Adaptive RT which dynamically adjusts RT windows every 3-4 seconds enabling narrow RT window lengths (down to 18 seconds) while minimizing missing data.
Challenge #5: Expediting method building and refinement strategies.
After identifying the target metabolites and lipids, researchers often struggle to determine the optimal instrument settings for the most sensitive, selective, and specific acquisition.
- Current state: Most of today’s triple quadrupole mass spectrometers have different ion sources relative to their discovery-based instrumentation. This requires additional method development, the need to optimize collision energy and dwell times per metabolite, and numerous replicate injections (when tied to online LC separation) or lengthy optimization steps (when using direct infusion of standards).
- A better solution: The Stellar mass spectrometer has similar ion sources to those of the Thermo Scientific Orbitrap-based mass spectrometers performing discovery experiments. The resulting set of metabolites and corresponding empirical data form spectral libraries used to automate targeted quantitative methods with the PRM Conductor tool in Skyline MS software.
The Stellar mass spectrometer can perform untargeted sample characterization using full scan MS. It can also perform gas phase fraction (GPF) leveraging the Stellar MS acquisition speed to create comprehensive spectral libraries using 4-5 replicate sample injections.
Conclusion: A smarter way to do translational metabolomics research
For more information, explore the Stellar mass spectrometer and download our new infographic.
*Glossary:
Higher-energy collisional dissociation (HCD): HCD is a fragmentation technique associated with Thermo Scientific Orbitrap instruments. Voltage offsets between components increase the kinetic energy of the precursor ion and dissociated in the ion routing multipole cell from collisions with neutral nitrogen molecules. After which they are transferred back to the C-trap and then injected into the Orbitrap mass analyzer for detection. Due to the increased kinetic energy, the resulting product ions also have increased kinetic energy and can undergo secondary and tertiary fragmentation that can result in a shift in more abundant low-mass product ions. HCD is associated with QQQ MS fragmentation performed in the Q2 cell or ion routing multiple cells in the Stellar mass spectrometer.
Collision-induced dissociation (CID) is a mass spectrometry technique to induce fragmentation of to improve selectivity, and specificity. For reference associated with ion traps, CID is defined as the method performed in the high-pressure linear ion trap using resonant RF to increase the kinetic energy of the selected ion which undergoes many ion-neutral collisions with helium over 3-5 ms resulting in fragmentation. Once product ions are formed, their m/z value is no longer resonant with the applied RF and are collisionally cooled back to the center of the ion trap. The primary difference between CID and HCD is the mass of the neutral collision gas (N2/Ar for HCD vs. He for CID) and the number of ion-neutral collisions during the process. As such, CID reduces the probability of secondary fragmentation and can alter the resulting product ion distribution as compared to HCD fragmentation.
Parallel reaction monitoring (PRM) is an ion monitoring technique based on performing either HCD or CID but measuring all resulting product ions in one scan event. The principle of this technique is comparable to SRM/MRM, but it is more convenient in assay development for absolute quantification as you can measure more signal from the targeted precusor ion and has been demonstrated to quantify down to attomole sensitivity in complex biological matrices.
Selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), is an important technique for quantitative analysis in complex matrices. SRM utilizes Q1 to isolate the precursor m/z value generally using a 1 Th isolation window, transferring the precursor to Q2 for HCD generation. The resulting product ions continue into Q3 where a diagnostic product ion m/z is isolated (1-2 Th isolation window) whose ion pathway continues to the detector. The SRM/MRM technique sets the instrument Q1/Q3 parameters (transitions) creating an ion beam in which the QQQ MS measures the resulting signal for a user-defined dwell time (1 to 100 ms). Researchers set two to four SRM/MRM transitions per analyte to increase the specificity and qualitative analysis of the resulting data. The SRM/MRM technique can achieve rapid, sensitive, specific quantification of the target protein in a complex system and excellent quantitative reproducibility. SRM/MRM has been identified as the gold standard for targeted quantitation for decades.