Stability measurements of antisense oligonucleotides by capillary gel electrophoresis.

The approach of using antisense oligonucleotides as potential drugs is based on hybridization of a short chemically-modified oligonucleotide with complementary cellular DNA or RNA sequences. A critical question is the stability of chemically modified antisense oligonucleotides in cellular environments. In a model system, resistance against various nucleases was evaluated by capillary gel electrophoresis (CGE). For some of the samples, matrix assisted laser desorption and ionization mass spectrometry (MALDI-MS) was used as an additional analytical tool to perform stability measurements. Using CGE, the enzymatic degradation of single nucleotides from the oligomer can be followed after different incubation times. 10% T polyacrylamide gels give baseline resolution for oligonucleotides ranging between 5 and 30 bases in length. The kinetic influence of a specific nuclease concentration and the antisense oligonucleotide structure on the cleavage reaction are discussed. Also, a simple desalting method to improve the injection efficiency and sensitivity of the method are described. Examples of measurements of chemically modified antisense 19-mers are presented.

Modification of a tunable UV-visible capillary electrophoresis detector for simultaneous absorbance and fluorescence detection: profiling of body fluids for drugs and endogenous compounds.

Using fused-silica optical fibres for fluorescence light collection and bandpass filters for selection of emission wavelengths, a capillary electrophoresis detection cell of a conventional, tunable UV-Vis absorbance detector was adapted for simultaneous fluorescence (at selected emission wavelength) and absorbance (at selected excitation wavelength) detection. Detector performance is demonstrated with the monitoring of underivatized fluorescent compounds in body fluids by micellar electrokinetic capillary chromatography with direct sample injection. Compared with UV absorption detection, fluorescence detection is shown to provide increased selectivity and for selected compounds also up to tenfold higher sensitivity. Examples studied include screening for urinary indole derivatives (tryptophan, 5-hydroxytryptophan, tyrosine, 3-indoxyl sulfate and 5-hydroxyindole-3-acetic acid) and catecholamine metabolites (homovanillic acid and vanillylmandelic acid) and the monitoring of naproxen in serum, quinidine in serum and urine and of salicylate and its metabolites in serum and urine.

Primary structure and function of novel O-glycosylated hirudins from the leech Hirudinaria manillensis.

Hirudin from the leech Hirudo medicinalis is a most powerful anticoagulant, and many isoforms have been described. In the present work, the primary structure of two hirudins from the leech Hirudinaria manillensis has been elucidated. The antithrombotic activity is similar to that of H. medicinalis hirudins although the sequence identity is below 60%. Surprisingly, the hirudins were found to be glycosylated at one site. Sugar analysis after methanolysis yielded fucose, galactose, and N-acetylgalactosamine. These results combined with data from matrix-assisted laser desorption ionization mass spectrometry, plasma desorption mass spectrometry, capillary zone electrophoresis, and lectin-binding tests indicate that the sequence is Fuc-Gal beta 1-3GalNAc-(O-threonine). This structure shows an interesting similarity to human blood group H determinants.

Analysis of synthetic peptides using matrix-assisted laser desorption ionization mass spectrometry.

Matrix-assisted laser desorption ionization mass spectroscopy (LDI MS), a novel method for analysis of large molecules, has been used for characterization of synthetic peptides and their by-products. The potential of LDI MS is demonstrated by analyzing crude synthetic peptides representing typical members of newly designed peptides and proteins. In the first case, a fragment condensation reaction yielding a highly hydrophobic six-helic bundle template-assembled synthetic protein (TASP) is monitored. Then, a crude 19-mer peptide designed to adopt an amphiphilic alpha-helical structure and its by-products from SPPS are identified. Finally, analysis of crude hirulog-1, a 20-mer peptide designed as a thrombin inhibitor, using C18 reversed phase high performance liquid chromatography (RP HPLC), capillary electrophoresis (CE) and LDI MS, manifests the potential of the latter method.

Fast protein sequence determination with matrix-assisted laser desorption and ionization mass spectrometry.

Measurement and identification of digested peptides by matrix-assisted laser desorption and ionization mass spectrometry (LDI-MS) is demonstrated. Synthetic human parathyroid hormone, pTH (1-34), with a molecular mass of 4117.8 Da was digested with carboxypeptidases Y and B and the sequence of 14 amino acids from the C-terminus of the peptide was determined by analyzing the molecular mass of the truncated peptides. Furthermore, a tryptic digestion of pTH (1-34) was carried out and a molecular mass map of pTH (1-34) was obtained. With the results of the proteolytic digestion a rapid confirmation of the amino-acid sequence of the protein was possible. It is shown that the results of the tryptic digestion can be used for the unambiguous identification of the amino acid residues Lys and Arg, which cannot be distinguished with a mass spectrometer because of their equal nominal masses. Several advantages of amino acid sequence determination by the combination of digestion and LDI-MS are obvious: high sensitivity in the low pmol range, fast digestion time due to high enzyme/substrate ratios, quantification is unnecessary because the amino acids are identified by their molecular mass differences, the low chemical expenditure for the digestions and the accuracy of the sequence determination. Measurements with LDI-MS are fast: sample preparation and the measurement take only a few min. The mass determination and amino acid sequence is completely unimpaired by amino acid contaminations or impurities in the sample. The sensitivity of the method is in the low pmol to fmol range and thus comparable to other analytical methods.

Isolation and purification of novel hirudins from the leech Hirudinaria manillensis by high-performance liquid chromatography.

The isolation and purification of novel hirudins from a crude extract of the leech Hirudinaria manillensis and their analytical characterization are reported. Initial purification by gel permeation chromatography on Sephadex G50 and anion-exchange chromatography on Q Sepharose fast-flow removed most contaminants and yielded a highly active extract. Two isohirudins (designated hirudin P6 and P18) were isolated and purified by successive reversed-phase high-performance liquid chromatography on silica-based stationary phases and anion-exchange chromatography on Mono Q. The final products were characterized by reversed-phase high-performance liquid chromatography, 252Cf plasma desorption time-of-flight mass spectrometry and capillary zone electrophoresis. The molecular masses determined by 252Cf plasma desorption mass spectrometry were 7416 dalton for hirudin P6 and 7199 dalton for hirudin P18.

Calibration of polyacrylamide gel columns for the separation of oligonucleotides by capillary electrophoresis.

Polyacrylamide-filled gel columns are used to separate oligonucleotide samples. For homopolymeric standard samples, plots of migration time versus molecular size are presented over a range of 30-160 bases. With 2.5-4% T and 3.3% C gels, good resolution over the examined mass range, with peak width at half height of 3 to 6 s, is obtained by applying electrical fields of 200-400 V/cm. The separation of heteropolymeric nucleotides by slab gel electrophoresis under routine conditions was compared with capillary gel electrophoresis. Using the same column and the same separation conditions, the plot of migration time versus base number is linear with an identical slope for three oligonucleotide samples which were examined, allowing a calibration of a gel-filled capillary for molecular mass determination.

Electrokinetic separation of chiral compounds.

Femtomole amounts of racemic mixtures of derivatized amino acids were resolved and analyzed rapidly in about 10 minutes by means of high-voltage zone electrophoresis with laser-fluorescence detection. The electrophoresis was performed in capillary columns containing a chiral support electrolyte. A number of dansyl amino acids were resolved by the diastereomeric interaction between the DL-amino acid and the copper(II) complex of L-histidine present in the support electrolyte. A combination of electro-osmotic and electrophoretic action caused all species, positively charged, neutral, and negatively charged, to pass through the 0.5- nanoliter detection volume where they were subjected to laser excitation.