NMR or MS – which technology to pick when analysing biofluids?

If you were to carry out metabolic profiling, the two most popular techniques used in metabolomics are Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). For the uninitiated, metabolomics is a method used in epidemiological studies to investigate metabolic factors that affect people’s health and their disease risk. Whether you want to carry out a targeted or untargeted analysis, both NMR and MS can be used to detect and identify metabolites and measure their concentrations accurately. However, both techniques have their own strengths and weaknesses.

The platform a researcher chooses primarily depends on the focus of their study and, of course, the nature of their samples. In this blog post, we list both the advantages and disadvantages of the two techniques. However, it is worth mentioning here that, in practice, employing multiple complementary technology platforms yields the most comprehensive results.

When to choose NMR?

NMR is a fast, cost-effective and versatile tool for metabolic profiling. Although, there are some drawbacks of using this technique as well. Here we look at both sides of the coin.

The Upsides of NMR

1. A powerful tool for metabolic profiling: NMR is quantitative, reproducible and does not require extensive steps for sample preparation, such as separation or derivatization [1]. The technique provides simultaneous quantification of routine lipids, lipoprotein subclass profiling with lipid concentrations within 14 subclasses, fatty acid composition and various low-molecular metabolites including amino acids, ketone bodies and gluconeogenesis-related metabolites in molar concentration units. Out of these, less than 20 biomarkers overlap with metabolomics platforms produced by MS. Meaning, NMR and MS are complementary when analysing these biomarkers.
2. A versatile tool for metabolomics research: From biomarker discovery to risk prediction and health tracking, NMR is suitable for all kinds of studies. Whether it is biomarker discovery, validation or translating metabolic information into practice, the technology supports all kinds of research and is regularly used in such studies. It has contributed to scientific discoveries in numerous areas including identifying genetic variants that affect individuals’ blood metabolic biomarkers and discovering novel biomarkers for chronic diseases.
3. Quick analysis: NMR analysis takes approximately 5 minutes since no sample preparation is needed. Analysing 10,000 samples takes about a month and 100,000 samples can be analysed in less than a year, when using one instrument. So, when you are using several instruments at the time, the analysis time reduces significantly.
4. Free of batch effects: The technique is free of ‘batch effects’ since the samples do not interact directly with the instrument [2].
5. Low-cost analysis: The NMR analysis costs less than the MS analysis. MS uses reference compounds for labelling which are considerably expensive. NMR doesn't use such compounds. So, sample preparation is minimal and there is no need for labelled standards, which makes the process cheaper.

The Downsides of NMR

1. A smaller number of biomarkers: Even though the number of biomarkers NMR analyses can detect is large, it remains smaller than what’s detectable using the MS technology.
2. Low sensitivity: NMR has lower sensitivity when compared with MS, which leads to detection of fewer biomarkers than MS. So, for certain applications like lipidomic, NMR alone may not provide sufficient resolution. However, the results can be improved, for instance, by performing multiple scans and using a higher magnetic field strength [3].
3. Bigger sample volume: NMR typically requires around 100 µL serum/plasma, which is higher than what an MS analysis demands.

When NMR is the best choice

Considering all the above-mentioned pros and cons, some of the best applications of NMR technology for medical research are as follows:

1. Biomarker discovery: Due to its high scalability and comprehensive coverage of multiple metabolic pathways, NMR is optimal for identifying biomarkers for chronic diseases. NMR can be applied to study large cohorts and the burden of multiple testing correction will not be too high.
2. Risk prediction: NMR can be used to build multi-biomarker risk prediction and stratification models for chronic diseases.
3. Omics integration: NMR is emerging as one of the most important platforms to complement genetics at a scale that is required for genome-wide association studies.
4. Clinical trials: NMR can be used to evaluate the fine-grained effects of therapeutic (and lifestyle) interventions in clinical trials. Since NMR doesn’t suffer from batch effects, it is the optimal technique for repeated measurements study settings.

When MS proves to be a good technique

Mass Spectrometry, or MS, is an extremely sensitive technology that can analyse a massive number of metabolites using a very small sample of blood. However, there are some inherent drawbacks of using this method too. Among others, it is a slow and expensive process.

The Upsides of MS

1. Powerful technique: Wide range of applications from identification to quantification of a variety of compounds in a sample.
2. Highly sensitive method: MS can readily quantify very low molecular concentrations, down to nanomolar and picomolar [1]
3. A large number of metabolites: When coupled with chromatography, both liquid chromatography (LC) and gas chromatography (GC), MS can detect hundreds and thousands of metabolites in a given sample.
4. Analysis of secondary metabolites: Chromatography, when coupled with MS, also allows one to analyse secondary metabolites even where the detection level is lower [1].
5. Small sample volume: When using the MS technology, it is possible to measure sample volumes as low as 10 µL serum/plasma.

The Downsides of MS

1. Batch effect: When chromatography is coupled with MS, the sample unavoidably interacts directly with the instrument. This, over time, leads to changes in measured analyte response [2].
2. Moderate reproducibility: Since the sample interacts with the instrument, it decreases its precision levels [1].
3. Sample recovery: While NMR allows using the same sample for multiple analysis, in MS, once a sample is analysed it can’t be recovered [3].
4. Slow analysis: Depending on the protocol used, analysis time can range from a few minutes and go up to one hour per sample, which makes it significantly slower than NMR.
5. Expensive analysis: MS requires sample preparation and separation (reagents as well as standards for metabolite identification) [2], which makes it a costlier technique than NMR.

When to use MS?

1. Metabolic profiling: MS is a good choice if the goal of the study is to obtain a very comprehensive coverage of the metabolome or when you need to do a quantitative analysis of a few metabolites.
2. Diagnosis of metabolic diseases: One should also pick MS to diagnose metabolic diseases utilising biomarkers with very low concentrations, or, when using well established standard routine protocol assay.

Combining NMR and MS

NMR and MS are highly complementary and can be used together to provide researchers with the opportunity to identify new metabolites and routinely measured and established biological pathways [2]. For instance, the recent integration of NMR and MS with pattern recognition models has helped to drive biomarker discovery. Combining these two technologies can also turn out to be more cost-efficient. Such combination approach would be ideal in a very large study where NMR could be first applied to identify the samples displaying most interesting results. These ‘extreme’ samples could then be analysed in more details using MS. Basically, NMR can be used for a quality screening of the samples, which can then be analysed further with MS. Another example of an intermixed application of the two technologies is when NMR lipoprotein analysis is combined with MS lipidomic.

Best of both worlds

Clearly, there is no winners or losers in this race. In fact, upon weighing all the advantages and disadvantages, one can conclude that there is no single analytical platform that can perform a complete quantification and identification of all molecules within a sample [1]. Therefore, more than one method must be employed for comprehensive metabolic profiling.

Comparing NMR to MS is like comparing SNP chips to sequencing. SNP chips are used to gather an overall view of a genetic variation which makes it optimal for discovery, whereas sequencing is optimal for fine-mapping genetic regions. Similarly, while NMR is a powerful technique for biomarker discovery, MS can be utilised for fine-mapping metabolic pathways. It, therefore, can be difficult to choose a single appropriate analytical strategy.

NMR offers some unique advantages and fills some deep holes in metabolomics research that cannot be filled by any other technology platforms. However, it should definitely not be viewed as the only tool for metabolomics. As it is also a vital and highly complementary tool for both liquid-chromatography mass spectrometry (LC-MS) and gas-chromatography mass spectrometry (GC-MS) based metabolomics [3].

References:

1. The strengths and weaknesses of NMR spectroscopy and mass spectrometry with particular focus on metabolomics research. Abdul-Hamid M. Emwas https://www.ncbi.nlm.nih.gov/pubmed/25677154
2. Systems level studies of mammalian metabolomes: the roles of mass spectrometry and nuclear magnetic resonance spectroscopy. Warwick B. Dunn, David I. Broadhurst, Helen J. Atherton, Royston Goodacre and Julian L. Griffin
   https://www.ncbi.nlm.nih.gov/pubmed/20717559
3. NMR Spectroscopy for Metabolomics Research. Abdul-Hamid Emwas, Raja Roy, Ryan T. McKay, Leonardo Tenori, Edoardo Saccenti, G.A. Nagana Gowda, Daniel Raftery, Fatimah Alahmari, Lukasz Jaremko, Mariusz Jaremko and David S. Wishart.
   https://www.ncbi.nlm.nih.gov/pubmed/31252628