The Analytical Needs of Neuroscience
What’s the best route to quantifying picomolar levels of neuropeptides in biological samples?
Ann Van Eeckhaut |
Seventy million people suffer from epilepsy worldwide, and with current drugs only controlling the symptoms (seizures) rather than curing the disease – and with more than 30 percent of patients resistant to current therapies – there is a clear need for more effective and better-tolerated options (1). To that end, we study neuropeptides (and their receptors), which are highly attractive in drug discovery because of their enormous diversity in functions and their involvement in almost every essential physiological process. Moreover, neuropeptides are preferentially released and exert their main actions when the nervous system is stressed, challenged or affected by disease. And because of their high receptor affinity and modulatory effect on neuronal communication, drugs interfering with peptidergic mechanisms are expected to be more potent and to give rise to less pronounced side effects compared with most currently available small-molecule therapeutics (2), (3).
Although progress has been made in the elucidation of the mechanisms of neuropeptide synthesis and the identification of neuropeptide receptors, physiological roles and mechanisms of release often remain elusive. To gain insight into central peptidergic effects, we can monitor the concentration changes of neuropeptides in the brain as a function of time; in such neurochemical studies, microdialysis is an established in vivo sampling technique that allows collection at basal extracellular levels as well as under pathological conditions. However, bioanalysis of the sampled neuropeptides is challenging because of the low concentration of these analytes in the brain (picomolar levels), the large number of potential interferences, and the limited sample volume (4). Although immunoassays are able to facilitate ultra-sensitive analyses, they suffer from a lack of standardization and a limited dynamic range and there can be large selectivity issues.
I believe that the high sensitivity and selectivity of nano (75-150 µm ID columns) UHPLC–ESI-MS/MS makes it the method of choice for the quantification of individual neuropeptides in brain dialysates. Over the years, nanoLC systems have become more robust, precise and user-friendly, but extracolumn peak broadening and long run times caused by significant gradient delays remain drawbacks. In my opinion, the availability of chip-based LC systems is a major step forward. Integrating all chromatographic components on a chip, including the electrospray emitter for MS detection, eliminates the need for fluidic connections. Consequently, delay and dead volumes as well as the post-separation volume are minimized, leading to a reduced total analysis time and decreased band broadening (5).
Independent of the analysis technique used, there are several challenges that should be taken into account when quantifying peptides, including analyte stability, the purity of the peptide standard, and solubility and related adsorption issues. Moreover, the behavior of neuropeptides under various sample preparation and chromatographic conditions is not well understood. In my experience, to obtain a method with maximal sensitivity, a tailored optimization – from peptide dissolution through to detection – should be performed for every peptide of interest. Indeed, because of the great diversity in peptide physicochemical properties, there really is no general approach. Fortunately, we can at least follow strategies based on design of experiments that can result in faster optimization of each method (6), (7).
As we want to quantify very low concentrations of peptides (low picomolar concentrations that represent attomole levels on column), the difficulties and challenges of analyzing peptides are even more pronounced; the adsorption of peptides is clearly more detrimental at this low level compared with micro- or nanomolar levels. In fact, aspecific binding of the peptides can occur at all steps of the method, from microdialysis sampling through to chromatographic analysis (7), (8).
Although improvements in MS technology are an important step forward for quantification of low concentrations of endogenous peptides, there is an additional challenge for analytical chemists: low sensitivity caused by the peptide ion current being divided amongst the multiple charged states that are commonly observed in ESI-MS. I believe in the capability of superchargers that can minimize the charge state distribution and thus maximize the relative abundance of one precursor ion, but more research is needed to assess their true potential.
The difficulty in quantifying endogenously released neuropeptides in vivo is evident in the low number of papers published. And it is even more difficult to find methods that were properly validated for the purpose. On many occasions I notice – to my regret – that analytical method validation is not always so well established in biomedical research labs. Whatever the field, we must remember that reliability of the generated data is the fundamental basis of scientific research.
- DF Weaver and B Pohlmann-Eden B, Epilepsia, 54, 80–85 (2013)
- T Hökfelt, T Bartfai, and F Bloom, Lancet Neurol 2, 463–472 (2003).
- S Kovac and MC Walker, Neuropeptides 47: 467–475 (2013).
- A Van Eeckhaut et al, Bioanalysis, 3, 1271–1285 (2011).
- A Van Eeckhaut and D Mangelings, J Pharm Biomed Anal, 133, 181-188 (2015).
- K Maes et al, J Chromatogr A, 1358, 1–13 (2014).
- K Maes et al, J Chromatogr A, 1360, 217–228 (2014).
- K Maes et al, Bioanalysis, 7, 605–619 (2015).