[Show abstract][Hide abstract] ABSTRACT: The synthesis and characterization of photocleavable peptide-DNA conjugates is described along with their use as photocleavable
mass marker (PCMM) hybridization probes for the detection of target DNA sequences by matrix-assisted laser desorption/ ionization
(MALDI) mass spectrometry. Three photocleavable peptide-DNA conjugates were synthesized, purified, and characterized using
HPLC and denaturing gel electrophoresis, as well as IR-MALDI and UV-MALDI. The hybridization properties of the conjugates
were also studied by monitoring their thermal denaturation with absorption spectroscopy. No significant difference in the
melting temperature (Tm) of the duplexes was observed between the unmodified duplex and the duplex in which one strand was modified with the photocleavable
peptide moiety. These conjugates were evaluated as hybridization probes for the detection of immobilized synthetic target
DNAs using MALDI-MS. In these experiments, the DNA portion of the conjugate acts as a hybridization probe, whereas the peptide
is photoreleased during the ionization/desorption step of UV-MALDI and can serve as a marker (mass tag) to identify a unique
target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed DNA/RNA analysis,
including gene expression monitoring, genetic profiling and the detection of pathogens.
Full-text · Article · Jan 2000 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Oligonucleotides containing a photocleavable biotin (5'-PC-biotin) were analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) with wavelengths in the ultraviolet (UV) and infrared (IR) from solution and after capture on streptavidin-coated agarose or magnetic beads. The analysis was used to monitor the release of the oligonucleotides as a result of photochemical cleavage of the biotinylated linker. Near-UV pulses (UV-MALDI) led to predominant release of the photocleaved product. In contrast, only the uncleaved analyte was detected using IR pulses (IR-MALDI). Results from MALDI analysis are also presented for DNA containing a photocleavable 5'-amino group which can be covalently linked to a variety of activated surfaces and marker molecules. In a demonstration of this approach, a 5'-PC-biotinylated 49 nt RNA oligonucleotide was enzymatically synthesized using a PC-biotin-r(AG) dinucleotide primer, captured on streptavidin coated magnetic beads and analyzed by UV-MALDI. Potential applications of photocleavable linkers combined with MALDI for the analysis of nucleic acids are discussed.
No preview · Article · Jan 2000 · Biomolecular Engineering
[Show abstract][Hide abstract] ABSTRACT: The 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-positional isomers of dihydroxybenzoic (DHB) acid were investigated with respect to their function as matrix-assisted laser desorption/ionization (MALDI) matrices. Optical absorption spectra of solid matrix samples, recorded in diffuse reflection from samples revealed peak broadening and a red shift of the peaks relative to the solution spectra. Single crystals of all isomers were grown from solution and analyzed by x-ray crystallography. Single crystals, as well as standard dried droplet and thin layer preparations with added cytochrome c, were analyzed by UV- (266, 308, 337, 355 nm) and IR- (2.94 μm) MALDI time-of-flight mass spectrometry (MALDI-TOF-MS). A spectrophotometric measurement of the heme absorption around 400 nm of redissolved single crystals showed a quantitative incorporation of the protein into crystals of 2,5-DHB and a partial protein incorporation with large statistical fluctuations into single crystals of 2,4-DHB. No protein incorporation above the detection limit of 10−5 molar analyte-to-matrix ratio was found for the other isomers. Best MALDI spectra from standard preparations were recorded for dried droplet preparations of 2,5-DHB at 337 and 355 nm and for 2,4-DHB at 266 and 308 nm. Both matrices performed well in the IR, too. 2,6-DHB yielded spectra of comparable quality at 337 nm and 2.94 μm, but only when prepared in a thin layer from an acetone solution. The results suggest that protein incorporation into the crystals of solid MALDI matrices is helpful, but not a prerequisite for MALDI. A large surface-to-volume ratio, typical for microcrystalline thin layer preparations supports protein desorption if no measurable incorporation occurs. Undesirable matrix adduct formation to the protein ions was seen for all DHB isomers at the wavelengths of 308 and 266 nm.
No preview · Article · Apr 1999 · International Journal of Mass Spectrometry
[Show abstract][Hide abstract] ABSTRACT: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) was used for the study of complexes formed by yeast seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) with tRNASer and tRNATyr. Cognate and noncognate complexes were easily distinguished due to a large mass difference between the two tRNAs. Both homodimeric synthetases gave MS spectra indicating intact desorption of dimers. The spectra of synthetase-cognate tRNA mixtures showed peaks of free components and peaks assigned to complexes. Noncognate complexes were also detected. In competition experiments, where both tRNA species were mixed with each enzyme only cognate alpha2.tRNA complexes were observed. Only cognate alpha2.tRNA2 complexes were detected with each enzyme. These results demonstrate that MALDI-MS can be used successfully for accurate mass and, thus, stoichiometry determination of specific high molecular weight noncovalent protein-nucleic acid complexes.
No preview · Article · Jan 1998 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: tRNATvr and tRNASer purified from bulk brewer's yeast tRNA were subjected to analysis by matrix-assisted laser desorption/ionisation time-of-flight
mass spectrometry. Choosing a mixture of 2,4,6- and 2,3,4-trihydroxyacetophenone and diammonium citrate as matrix a mass resolution
of up to 220 (FWHM) was achieved in the linear mode of operation. Cation adduct suppression by addition of cation exchange
beads and a chelating agent (CDTA) is shown to substantially improve mass resolution for this class of molecules.
Full-text · Article · Jun 1997 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: The determination of RNA sequences using base-specific enzymatic cleavages is a well established method. Different synthetic
RNA molecules were analyzed for uniformity of degradation by RNase T1, U2, A and PhyM under reaction conditions compatible with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS),
to identify the positions of G, A and pyrimidine residues. In order to get a complete set of fragments derived from cleavage
at every phosphodiester bond, the samples were also subjected to a limited alkaline hydrolysis. Additionally, the 5′-terminus
fragments of a 49mer RNA transcript were isolated by way of 5′-biotinylation and streptavidin-coated magnetic beads (Dynal),
followed by a RNase U2 digestion. MALDI-MS of the generated fragments is presented as an efficient technique for a direct read out of the nucleotide
Full-text · Article · Jun 1997 · Nucleic Acids Research