Sensitive & Selective Detection of Neuroactive Compounds

Peptides

Since 19891, the group has worked on the development of strategies for the determination of peptides and other neuroactive substances in biological samples. We were motivated by the recognition of two things:

Peptides are most commonly determined by immunoassay. Immunoassay relies on the specificity of antibodies for the epitopic region of the target molecule. As the figure below shows, the interface between the antibody (grey, red and blue balls) and the target epitope (green balls) is only a small region.

http://www.biology.arizona.edu/immunology/tutorials/antibody/structure.html
 

Peptides are synthesized as prepropeptides which are cut by specific enzymes into propeptides, then into active peptides. Ultimately, peptides are then inactivated by hydrolysis typically by ectoenzymes in the brain. These are membrane-bound, outward facing hydrolytic enzymes. What this means is that a propeptide, the active peptide, and an inactive form may all have the same epitope. The figure below represents the primary sequence of several propeptides with the active regions labeled and shaded.   Note that, in the top sequence of proopiomelanocortin, the pentapeptide met-enk (YGGFM), itself a bioactive peptide, is contained in the propeptide, in b-melanocytestimulating hormone (b-MSH) and in b-endorphin. While immunoassays are unquestionably valuable, in the case of peptides, a separate-and-detect approach is preferable. The problem, of course, is that immunoassays can be very sensitive. So we seek a sensitive and selective method involving separations.

The biuret reaction

We use the reaction of Cu(II) in basic solutions to derivatize peptides following their separation by HPLC (reversed phase).

As the scheme shows, the amide protons dissociate, and stable complexes with three nitrogen and one oxygen donor (NNNO) or four nitrogen donors (NNNN) are formed. As Dale Margerum showed in a series of papers in the ‘70s and ‘80s, these complexes are electroactive. We have extended those studies, having worked on over 30 bioactive peptides from dipeptides to insulin2-8

The postcolumn reaction involves the addition of Cu(II) in basic copper tartrate after the separation. Particularly with microcolumn separations, it is important to avoid adding bandspreading. As we show below, we can mix effluent and reagents effectively.

Postcolumn reactors in microcolumn separations

Using a new technology 9-13, we have prepared postcolumn mixer/reactor (below). The reaction product is detected with an electrochemical detector, typically a carbon fiber in the capillary outlet.  The ‘lab on a chip’ community14 has developed clever approaches to on-chip reagent mixing that obviate the problems posed by diffusional mixing in laminar flow.  However, we are convinced that for microcolumn liquid chromatography Taylor dispersion is a superb and simple way to mix reagents.

We have developed a simple method to assess extracolumn bandbroadening and we have applied it quantitatively to the postcolumn mixing problem15. We use a large, 1 mL, injection, and then differentiate the signal from it (below). 

 

 

The widths of the resulting peaks are completely due to bandspreading – there is no contribution from the intrinsic width of the injection as that is zero.  For the mixer shown above and many like it (n=7) we cannot distinguish between a mixer and an equal length of 50 mm tubing.  Thus the confluence of the streams adds nothing measurable to bandspreading.

Methods

Along the way, we and our collaborator Mats Sandberg have developed methods for some specific species.

S-Sulfocysteine

S-sulfocysteine is a biological mimic of glutamate.  Its EC50 for the NMDA receptor is actually lower than that for glutamate.  Cysteine is neurotoxic.  We hypothesized that the oxidation of cysteine to S-sulfocysteine was the mechanism of the toxicity.  There were no methods good enough, so we developed one using HPLC with electrochemical detection.  We found no significant SSC in hippocampus extracts following exposure to toxic levels of cysteine (< 20 nM which is << EC50).  Thus, this mechanism of cysteine toxicity is ruled out.

N-acetyl Aspartate

A new method has been developed for determination of N-acetyl-aspartate, a neuronal aerobic metabolism marker.  This functionally enigmatic amino acid is present in high concentrations in brain and almost exlusively in neurons. The concentration of N-acetyl-aspartate shows a good correlation to cognitive function in humans and is produced in mitochondria. It is thus considered as a marker of neuronal metabolic aerobic activity.

Enkephalins

We are in the process of validating an HPLC method for leu-enkephalin (YGGFL, Tyr-Gly-Gly-Phe-Leu) and its hydrolysis product GGFL (Gly-Gly-Phe-Leu).  Our current system using a 100 mm ID reversed phase column, an acetonitrile/water/1% trifluoroacetic acid gradient, postcolumn addition of biuret reagent and a single carbon fiber electrode as the detector at 0.7 V vs Ag/AgCl.

Some peptides have a structure that influences its reactivity with Cu(II). One such class is the chemotactic peptides which have a formylated amine terminus.  As the nucleophilicity of these peptides is low, we expected and found that the reaction rate is low.  A complete investigation 16 showed that the Cu(II) has two routes to formation of the biuret complex, one via the amine terminus and one via the caboxylate terminus.  The latter is the important path for N-formyl- and N-acetyl peptides. 

We discovered a dramatic effect.  Bicarbonate accelerates the reaction at the carboxy terminus.  With proper attention to temperature and solution composition, the N-blocked peptide N-f-MLF will react to a significant degree in only a few seconds (kobs = 0.24 s-1).  With a standard borate buffer at room temperature, the rate of reaction is 1000 time slower (kobs = 2.5 x 10-4 s-1), rendering these peptides undetectable.

Another set are TRH and related peptides. The his-containing peptides react with Cu(II) readily, however the H2E and H2F derivatives do not17, 18. These are the only peptides that we have discovered that are not reactive.

 

Detection

We have made advances in electrochemical detector design and in “Photoluminescence following electron transfer” (PFET), a technique that can have the selectivity of electrochemistry with the sensitivity of fluorescence.

The idea behind PFET is simple.  Instead of oxidizing a species in an electrochemical detector, we use a special, dissolved oxidant to oxidize it.  The oxidant is not fluorescent, but its reduced form is.  Detection is based on measuring the fluorescent signal (below) that arises from the conversion of nonfluorescent oxidant to the fluorescent product.  Ru(bpy)33+ was the first oxidant (Ru(bpy)32+ is fluorescent) used for PFET.  It has been applied to the determination of dynorphin A and its fragments4, 7, 9, 10, 16, 19-21. In this application, we do not employ copper, rather we use the inherent redox activity of Tyr and Trp. 


We have expanded the application of Ru(bpy)32+ to detect reductants created electrochemically, and have applied that to the determination of explosives.21 The idea is shown schematically below:

TNT 

-->

TNTred + ne-

(1)

TNTred+ nRu(bpy)33+

-->

nRu(bpy)32+  + X

(2)

 

Here the nonfluorescent reagent Ru(bpy)33+ , generated online electrochemically, is reduced by the product of the electrochemical reduction of the nitrocompounds (FCA is a ferrocene derivative that we use as a one-electron standard).  The Ru(bpy)32+ formed is quantitatively related to the original concentration of analyte.  A chromatographic separation and detection of explosives near the detection limit (which is better by x10 than any other published method) is shown in above.

Recently, we have developed the use of the less oxidizing Os(bpy)32+. We have successfully applied this to the determination of catecholamines in microdialysate22-24.

Publications

  1. Warner, A. M.; Weber, S. G. Analytical Chemistry 1989, 61, 2664-2668.

  2. Chen, J. G.; Sandberg, M.; Weber, S. G. Journal of the American Chemical Society 1993, 115, 7343-7350.

  3. Chen, J.-G.; Weber, S. G.; Glavina, L. L.; Cantwell, F. F. Journal of Chromatography 1993, 656, 549-576.

  4. Chen, J.-G.; Woltman, S. J.; Weber, S. G. Journal of Chromatography, A 1995, 691, 301-315.

  5. Chen, J.-G.; Vinski, E.; Colizza, K.; Weber, S. G. Journal of Chromatography, A 1995, 705, 171-184.

  6. Chen, J.-G.; Weber, S. G. Analytical Chemistry 1995, 67, 3596-3604.

  7. Woltman, S. J.; Chen, J.-G.; Weber, S. G.; Tolley, J. O. Journal of Pharmaceutical and Biomedical Analysis 1995, 14, 155-164.

  8. Chen, J.-G.; Logman, M.; Weber, S. G. Electroanalysis 1999, 11, 331-336.

  9. Woltman, S. J.; Even, W. R.; Sahlin, E.; Weber, S. G. Analytical Chemistry 2000, 72, 4928-4933.

  10. Sahlin, E.; Beisler Amy, T.; Woltman Steven, J.; Weber Stephen, G. Analytical chemistry 2002, 74, 4566-4569.

  11. Sahlin, E.; Weber, S. G. Journal of Chromatography, A 2002, 972, 283-287.

  12. Sahlin, E.; ter Halle, A.; Schaefer, K.; Horn, J.; Then, M.; Weber Stephen, G. Analytical chemistry 2003, 75, 1031-1036.

  13. Beisler, A. T.; Sahlin, E.; Schaefer, K. E.; Weber, S. G. Analytical Chemistry 2004, 76, 639-645.

  14. Reyes, D. R.; Iossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002

  15. Beisler, A. T.; Schaefer, K. E.; Weber, S. G. Journal of Chromatography, A 2003, 986, 247-251.

  16. Shi, F.; Woltman, S. J.; Weber, S. G. Analytica Chimica Acta 2002, 474, 1-9.

  17. Meng, R.; Xia, W.; Sandberg, M.; Stephens, R.; Weber, S. G. Journal of Chromatography, A 2005, 1071, 179-184.

  18. Meng, R.; Becker, J.; Lin, F.-T.; Saxena, S.; Weber, S. G. Inorganica Chimica Acta 2005, 358, 2933-2942.

  19. Woltman, S. J.; Alward, M. R.; Weber, S. G. Analytical Chemistry 1995, 67, 541-551.

  20. Xia, W.; Woltman, S. J.; Sandberg, M.; Weber, S. G. Journal of Electroanalytical Chemistry 1997, 435, 39-46.

  21. Woltman, S. J.; Even, W. R.; Weber, S. G. Analytical Chemistry 1999, 71, 1504-1512.

  22. Jung, M. C.; Weber, S. G. Analytical Chemistry 2005, 77, 974-982.

  23. Jung, M. C.; Munro, N.; Shi, G.; Michael, A. C.; Weber, S. G. Analytical Chemistry 2006, 78, 1761-1768.

  24. Jung, M. C.; Shi, G.; Borland, L.; Michael, A. C.; Weber, S. G. Analytical Chemistry 2006, 78, 1755-1760.