A Tiny Sample Problem
A novel CE-MS technique shows great promise for sample-restricted metabolomics.
Metabolomics is a key discipline in studying molecular and cellular processes in living cells and organisms, with the ultimate aim of getting answers to biological and clinical questions. Today, state-of-the-art analytical techniques, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) coupled to liquid chromatography (LC) or gas chromatography (GC), provide global and reproducible profiling of (endogenous) metabolites in biological samples.
Regardless of important innovations in analytical technology realized over the past decade, the current analytical toolbox still has difficulty analyzing ultra-small biological samples. Therefore, a significant number of crucial biomedical and clinical questions remain unanswered. For example, the analysis of (trace-level) metabolites in volume/material-limited biological samples, such as liquid biopsies, cerebrospinal fluid (CSF) from mice, cancer stem cells or even a single cell, remains a tremendous analytical challenge. To enable “sample-restricted” metabolomics, new miniaturized analytical workflows and technologies are needed urgently.
Over the last few years, a number of research groups have developed miniaturized analytical techniques for metabolomics studies of restricted sample amounts, proving that research in this particular field is very active (1–3). My own research is focused on the same goal, in particular using capillary electrophoresis (CE) because it is well suited for analyzing minute amounts of sample. Moreover, analytes are separated by their charge-to-size ratio making CE an attractive tool for profiling highly-polar and charged metabolites.
When it comes to coupling CE to electrospray ionization (ESI)-MS, I have used a novel sheathless interface design (invented by Mehdi Moini, George Washington University, Washington DC, USA) (4), which allows full exploitation of the intrinsically low-flow property of CE to significantly improve sensitivity and reduce ion suppression. The developed CE-MS platform has been used for highly sensitive analysis of ionogenic compounds in various biological samples (5, 6). A volume of only 2 μl was required in the sample vial for up to 10 consecutive injections of about 9 nl into the CE-MS platform, demonstrating the potential of the approach for analyzing volume-limited biological samples. For example, mouse CSF, of which only a few microliters are obtained under proper experimental conditions, could be analyzed directly with the CE-MS platform using minimal sample pretreatment (only 1:1 dilution with water). The approach retains sample integrity, which is essential for comprehensive profiling of polar and charged metabolites. I expect that the proposed technology will also offer a unique chiral perspective on the composition of ultra-small biological samples as the suppression effect of chiral selectors, such as cyclodextrins, may be limited under low-flow ESI-MS conditions.
In the forthcoming Microscale Separations and Bioanalysis symposium (April 3–7, 2016), to be chaired by Philip Britz-McKibbin, special attention will be devoted to key developments in analytical technologies and workflows for sample-restricted biological/clinical problems. I’ll be co-chairing the “Comprehensive Omics” session with Oleg Mayboroda (Leiden University Medical Center), which has the aim of providing the separations science community with a representative glimpse of recent achievements and innovations, as well as an interactive forum for discussing and exchanging ideas.
Overall, my view is that recently developed miniaturized analytical technologies appear to provide very promising results for sample-restricted metabolomics studies. But the next important step for these approaches is to show merit in large-scale biological and clinical studies. Such data are critical to promote these approaches further as a potential diagnostic tool. Additional work is also necessary on the pre-analytics side to effectively handle volume/material-limited biological samples and to efficiently transfer the analytes into the nanoscale analytical separation technique. Despite many analytical challenges in front of us, I anticipate that further development in this research field will have a major impact in metabolomics and bioanalysis in general as the new technologies continue to lead us in new directions, which is to say, towards a deeper understanding of biological functions in sample-restricted cases.
- P Kiefer et al., “Nanoscale ion-pair reversed-phase HPLC-MS for sensitive metabolome analysis”, Anal Chem, 83(3), 850–855 (2011). PMID: 21166460.
- P Nemes et al., “Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry”, Nat Protoc, 8(4), 783–799 (2013). PMID: 23538882.
- KT Myint et al., “Quantitative profiling of polar cationic metabolites in human cerebrospinal fluid by reversed-phase nanoliquid chromatography/mass spectrometry”, Anal Chem, 81(3), 1121–1129 (2009). PMID: 19125563.
- M Moini, “Simplifying CE-MS operation. 2. Interfacing low-flow separation techniques to mass spectrometry using a porous tip”, Anal Chem, 79(11), 4241–4246 (2007). PMID: 17447730.
- R Ramautar et al., “Enhancing the coverage of the urinary metabolome by sheathless capillary electrophoresis-mass spectrometry”, Anal Chem, 84(2), 885–892 (2012). PMID: 22148170.
- R Ramautar et al., “Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS”, Anal Bioanal Chem, 404(10), 2895–2900 (2012). PMID: 23052875.
Assistant Professor, Leiden Academic Center for Drug Research, Division of Analytical Biosciences, Leiden University, The Netherlands.
