Vertiefungskurs Combinatorial Chemistry

Sommersemester 2000

Experiment 4:  MALDI analysis of crude

Wednesday, May 31, 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.  Assuming that you have succeeded in coupling the carboxylic acids you selected to the amino-terminal DNA, and that the deprotected product of that reaction can be detected via MALDI-TOF mass spectrometry, you will have the pleasure of seeing the first spectrometric evidence for the new compounds you have made.  The experiment is straightforward, in the sense that you will only have to prepare the samples, and your teaching assistant will acquire the spectra for you.
A few words about MALDI-TOF mass spectrometry.  First of all, its acronym stands for Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight mass spectrometry.  It is a relatively new technique, having been first described only a dozen year ago (Karas, M. & Hillenkamp, F. (1988). Laser desorption ionization of proteins with molecular mass exceeding 10000 Daltons. Anal. Chem.  60, 2299-2301.).  It is one of the two new mild desorption techniques (the other being electrospray mass spectrometry, ESI) that allows desorbing polar and fragile biomacromolecules into the gas phase.  One great advantage over ESI is that the singly charged ions are formed predominantly with MALDI.  As a result, one sees one sharp, predictable peak that predominates: that of the pseudomolecular ion (M+H or M-H).  Depending on the concentration, there will also be less prominent peaks for the doubly charged ions, flying at half the m/z of the singly charged ion (e.g. [M-2H]2-).  Some additional information on MALDI and ESI can be found in: Burlingame, A. L., Boyd, R. K. & Gaskell, S. J. (1996). Mass spectrometry. Anal. Chem. 68, 599R-651R; or Fitzgerald, M.C. & Siuzdak, G. (1996). Biochemical mass spectrometry: worth the weight? Chemistry & Biology 3, 707-715.  You may also find our very first little paper on this issue entertaining: Sarracino, D.; Richert, C. Quantitative MALDI-TOF spectrometry of oligonucleotides and a nuclease assay. Bioorg. Med. Chem. Lett. 1996, 6, 2543-2548.
In order to allow for mild desorption, your analyte will have to be incorporated in a matrix of a small molecule that absorbs the laser shots and helps to produce the singly charged ions.  These are usually derivatives of benzoic acids or relatively acidic phenols.  In your case this will be 2,4,6,-trihydroxyacetophenone, a phenol derivative.  The molar ratio between analyte and matrix should be in the range of 1:1'000 to 1:10'000.  MALDI is very sensitive, and 20 pmol of your compound on the target plate should give an easily detectable signal.  One problem that plagues MALDI is the formation of metal ion adducts.  In order to become singly charged, the oligonucleotide can either have its phosphodiesters protonated or associated with sodium or potassium cations.  The latter leads to a splitting of the main signal into signals of the general formula MHn, MHn-1Na1, MHn-2Na2, etc.  To prevent this, an excess of ammonium salts is added.  In the resulting equilibrium, these displace sodium and potassium ions and form the ammonium salt of the DNA.  When desorbed in the MALDI instrument, these ammonium salts decompose into the protonated DNA and ammonia, which is a gas and is pumped off.  Thus, the protonated form of the oligonucleotide is obtained almost exclusively.
One word of caution.  You will probably see a series of peaks in the spectra you get, hopefully dominated by the peak for your target compound, but certainly containing peaks of failure products from the synthesis.  The failures may look more prominent than they really are, because longer oligonucleotides are desorbed/ionized more poorly in the MALDI spectrometer than shorter ones.  In short:  the shorter ones fly better (Schneider, K.; Chait, B. T. Org. Mass Spectrom. 1993, 28, 1353.)  You may also observe that the intensity of the failure peaks may be more intense in the spectra of some products than in others, even though they were synthesized on the same DNA.  This may have two main reasons.  One is that the carboxylic acid residue you have appended does influence the desorption/ionization propensity of your DNA to a different extent.  Acid residues that are large and polar or induce aggregation are more likely to depress the signal of your target compound than small and unfunctionalized ones.  Further, the carboxylic acid residue you have introduced may lead to fragmentation of the DNA portion of your hybrids during the deprotection.  Several possible attacks are possible, particularly if your acid residue contains nucleophilic groups that can attack the phosphotriesters initially available during the deprotection (the phosphodiester anions are more kinetically inert).  I recommend that you try to analyze the fragments carefully and try to assign them to molecular species that could have formed during the synthesis or deprotection reaction.  We will try to remove excess failures in your crude products during next weeks experiment ("Pre-Purification").
Enjoy this week's lab!

Analysis of crude oligonucleotide hybrids via MALDI-TOF mass spectrometry.  The matrix conditions chosen are modified from: Pieles U., Zürcher W., Schär M. & Moser H.E. (1993). Nucleic Acids Res.  21, 3191-3196.
 When you enter the lab, your teaching assistant will have performed the deprotection reactions (treatment with 500 µL saturated aqueous ammonia solution or "ammonium hydroxide" for 16h) and will have opened the reaction vessels to allow excess ammonia to escape (overnight).  You will be left with a mixture consisting of the supernatant containing your crude product and the protecting group fragments in aqueous solution and the cpg at the bottom of the flask.  Please siphon off the supernatant carefully with a syringe with a small gauge needle and wash the cpg twice with 50 µL of water.  The combined aqueous solutions can now be directly sampled and the aliquot drawn used for MALDI-TOF analysis.  Assuming that you used 1 mg of the cpg with approximately 30 nmol of C*GGTTGA (where C* stands for the 5'-amino-5'-deoxycytidine residue) and that you have 10% of your product (a conservative figure, I hope) you will have 3 nmoles in 600 µL.  If you take 1 µL of the resulting solution, you will have approximately 5 pmols of your compound.  So, you should draw a sample of 1 µL from every solution of your crude products and transfer it into a separate Eppendorf cup.  To this, you should add 3 µL of a solution of THAP (0.3 M in ethanol) and 2 µL of a solution of diammonium hydrogencitrate in water (0.1 M).  Both of these solutions will be provided by your teaching assistant.  Now, you should mix the solution by aspiring and expelling the solution a few times with the "pipetman".  Of the resulting solution you will now spot 1 µL on one of the numbered areas on the stainless steel target for MALDI MS that will be brought by your teaching assistant.  It is important that you write down which areas you are using, so that there will be no confusion when the target plate is being analyzed.

After having prepared your samples for MALDI analysis, please keep the remaining 599 µL of your crude samples!  These should be submitted for lyophilization in their current form (please make sure they are well labeled with a label that cannot come off).  After lyophilization, next week, the residue will be taken up in water (50 µL) and fractions of all solutions will be pooled, so that we can perform a prepurification of the library prior to performing selection experiments.

A Note at the End.  We regret that the last experiment took longer than expected.  We hope that this week's experiment will be much shorter.  Of course, you can leave after having prepared the MALDI spot, if you do not want to wait around for the acquisition of the MALDI spectra.  Your teaching assistant will provide you with the spectra next week.