Experiment 6: Quantitation of Product
Wednesday, June 14, 2000
Location: You will meet in room Z 744 for this week's experiment
(Verfügungsgebäude, 7th floor level).
Teaching Assistant: Your teaching assistant for this experiment
is Andriy Mokhir.
Information on the experiment.
The title of today's experiment is "quantitation of product". Now, of course, assuming that you have made the required four modified oligonucleotides, it should really be "quantitation of products". Since your time is limited, you are expected to quantify only one of your products and estimate the quantity of the others. During the last few days, Andriy Mokhir and Jason Mayo have finished the deprotection of your crude products (in some cases a new mixed coupling had to be performed), prepared injection solutions for HPLC, injected the pooled crudes of your modified oligonucleotides, and acquired MALDI spectra of samples from the fractions collected. This has led to the pre-purification of most of your compounds. Further, Andriy and Jason have prepared stock solutions of the pre-purified compounds, so that you can perform today's experiment. I hope you will find the time to thank them for their very substantial efforts. There are two parts to today's experiment. One is to determine the concentration of one oligonucleotide hybrid via UV-vis spectrophotometry, and the other is to produce solutions for a calibration experiment for quantitative detection of your hybrid via MALDI-TOF mass spectrometry.
There is no need to explain how one quantifies a compound via UV-vis, using Lambert-Beer's Law, so I will just mention that since you have made small quantities of compounds, I hope that you will be able to see a UV signal, and also that I hope you will be able to recover the material by lyophilizing the solution after the UV-spectrum was taken. The lmax for the main absorbance of the nucleobases is at approximately 260 nm. The absorbance of an oligonucleotide in single-stranded form is in a good approximation the linear combination (sum) of the extinction coefficients of the nucleobases that it contains. The extinction coefficients of these are known. We usually work with: e = 8800 M-1cm-1 for T, 7300 M-1cm-1 for C, 11700 M-1cm-1 for G, and 15400 M-1cm-1 for A (see e.g. Brown, T. and Brown, D.J.S. in Eckstein, F. (ed.) Oligonucleotides and Analogues, A Practical Approach. IRL Press, Oxford: 1991, pp. 20).
Quantitative MALDI is a less traditional technique. Absolute peak intensities do not mean much in MALDI-TOF mass spectrometry, since signal intensity depends exquisitely on the localization of the laser beam on the semicrystalline matrix. Mass spectrometrists also speak of "sweet spots" when referring to those locations in the matrix "cake" where strong analyte signals can be obtained. To allow for quantitation of a compound, one has to employ an internal standard, i.e. a known quantity of a compound that is chemically similar to the analyte, so that relative, rather than absolute, signal intensities can be determined. If an appropriate internal standard is used (in your case a slightly longer, unmodified oligonucleotide) and near exhaustive ablation conditions are employed in the spectrometer (high laser power, averaging over many laser shots and spectra), reasonable quantitation results can be obtained (see e.g. Sarracino, D. & Richert, C. Bioorg. Med. Chem. Lett. 1996, 6, 2543) The results also depend on the matrix chosen, but we do not have to go into these details.
The goal of this part of the experiment is to prepare a dilution series, i.e. a series of solutions with decreasing concentrations of the oligonucleotide hybrid in question, and to acquire MALDI spectra of these with a fixed amount of the internal standard in the matrix preparation. One should then see a decrease in the relative peak intensity (analyte/internal standard). Plotting concentration against relative peak intensity should yield data points to which a straight line can be fitted. We are planning to cover one order of magnitude in concentration with the experiment planned, which is sufficient for monitoring the selection experiments that are supposed to be performed in one of the next labs. Some specific recommendations for today follow.
Determining product concentration via UV spectrophotometry.
You will obtain stock solutions of the pre-purified compounds with a volume
of 10 µL. At the same time, you will receive a copy of the
HPLC chromatogram of your library of pooled crude oligonucleotide hybrids.
Since the detection in the HPLC is also via UV, one can quantify the compounds
eluted (as long as they are reasonably well separated) via the integration
of their corresponding HPLC peaks. Conveniently, you will also obtain
a collection of copies of MALDI spectra acquired off of the fractions from
the HPLC run. Please choose the stock solution of the compound that
gave the most intense HPLC peak. Of the 10 µL of stock solution
that you have, 5 µL should be taken and diluted with 495 µL
of deionized water. Of the resulting solution of 500 µL a UV
spectrum in the wavelength range of 230-400 nm should be acquired with
the help of your teaching assistant. Please make sure you rinse the
semimicro UV-cuvette used prior to and after the acquisition of your spectrum,
so that there is no cross-contamination. After having the spectrum
acquired, please recover the solution of your compound (do not scratch
the cuvettes with glass pipettes!, pouring only), label it carefully and
give it to your teaching assistant for lyophilization.
Determine the concentration of your analyte in the stock solution, using the extinction coefficient to be calculated from the values given above, and also determine the absolute amount of compound obtained, together with the yield, assuming a loading of 30 nmol of DNA per mg of cpg and a yield for the DNA synthesis of 50%. If you happen to have a substituent on your DNA that contributes substantially to the UV-absorbance at 260 nm, you have to add this to the extinction coefficient of the DNA portion of the hybrid. If you do not know the extinction coefficient from the literature, you will have to determine an approximate extinction coefficient by acquiring a spectrum of the carboxylic acid building block used in CH2Cl2. I guess you know how to produce a solution of defined concentration for this (a few mg in 10 mL methylenechloride will work in most cases).
Preparing Samples for MALDI Calibration Experiment. Using
only a fraction of the remaining 5 µL of your stock solution (you
will need the rest for the selection experiments), a dilution series has
to be prepared. For this, please transfer 1 µL of this solution
to a new small Eppendorf cup and dilute it with 9 µL of deionized
water. Of the resulting 10 µL solution, the following aliquots
should be put into one small Eppendorf cup each: 5 µL, 2.3 µL,
1.1 µL, and 0.5 µL. (The excess is required to ensure
that you do not "run dry", due to loss based on adsorption on surfaces
and inaccuracies in the pipetting). The Eppendorf cups containing
each of these aliquots should again be labeled carefully (make sure you
include at least your initials, if they are not identical to any other
student, an identifier for the compound, and the dilution information).
After lyophilization, the samples will be taken up by your teaching assistant
in an equal amount of matrix mixture containing the internal standard.
A fifth sample will be prepared with the same matrix mixture but without
any analyte to measure any background that may exist.