Biomedical subjects

F Hillenkamp

Publications and source records attributed to F Hillenkamp.

At least 19 recordsLinked to original sources

PubMed2001

Fluorescent DNA: the development of 7-deazapurine nucleoside triphosphates applicable for sequencing at the single molecule level.

7-Deaza-2'-deoxyadenosine and -guanosine phosphoramidite building blocks as well as corresponding 5'-triphosphate derivatives are described carrying in position 7 substituents such as iodo, hexyn-1-yl or 5-aminopentyn-1-yl residues. The phosphoramidites were used to synthesize a series of modified oligodeoxynucleotides. A systematic study of the thermal stabilities of these oligonucleotide duplexes demonstrated that the 7-substituents are well accommodated in the major groove of B-DNA. The 7-(aminoalkyn-1-yl)-7-deazapurine 2'-deoxynucleoside triphosphates were labeled with bulky fluorophores such as Rhodamine Green(R) or tetramethylrhodamine.

Biochemistry↗

PubMed2001

Localization of analyte molecules in MALDI preparations by confocal laser scanning microscopy.

In this study, the incorporation of Texas Red-labeled avidin into crystals of 2,5-dihydroxybenzoic acid (2,5-DHB) and 2,6-DHB (used as matrixes for matrix-assisted laser desorption/ionization (MALDI)) was investigated by fluorescence spectrophotometry and confocal laser scanning microscopy (CLSM). The analyte distribution in crystals, grown slowly under controlled conditions, was compared to the analyte localization in different standard preparations (dried-droplet and thin-layer preparation). Texas Red turned out to be a useful fluorescence label in the acidic environments of typical matrixes. Earlier results by absorption spectrophotometry could be confirmed by fluorescence measurements; 2,5-DHB incorporates the analyte proportionally, while 2,6-DHB excludes the protein from its crystal lattice. It is found that the analyte distribution can be analyzed well in both single crystals and standard preparation, by CLSM using Texas Red-labeled analytes. The present study allows for a conclusive and consistent interpretation of analyte incorporation into MALDI preparations.

Avidin↗

PubMed2001

Fragmentation mechanisms of oligodeoxynucleotides studied by H/D exchange and electrospray ionization tandem mass spectrometry.

We used solution-phase hydrogen/deuterium (H/D) exchange and multistage tandem mass spectrometry (MS/MS) experiments in an electrospray ion-trap mass spectrometer operating in the negative-ion mode to investigate the consequences of the loss of a high proton-affinity (PA) base from T-rich tetra and hexadeoxynucleotides. The T-rich oligodeoxynucleotides containing one or two other nucleobases take advantage of the mass spectral inertness of T because fragmentation of a T-rich oligomer is simple, allowing a tight focus on those processes of interest. Furthermore, determination of T-rich oligodeoxynucleotides may be a starting point in the development of a mass spectrometric scheme to understand the mutagenicity of various types of DNA damage by UV radiation. For nine oligodeoxynucleotides, the nucleobases were charged by nearly exclusive D transfer and then expelled as neutral bases. Loss of the base located at the 3' end is preferred over that from the 5' terminus when the two bases are identical. The observation of partially exchanged fragments from a completely exchanged precursor ion proves intramolecular H/D exchange between hydrogen atoms that can exchange in water and those that cannot. The multiplicity of the product-ion peaks provides information on decomposition pathways and origins of the product ions and shows that the loss of base is the first step in all fragmentation of hexanucleotides, but is a competitive process for tetranucleotide fragmentation.

Deuterium↗

PubMed2001

Metastable decay of negatively charged oligodeoxynucleotides analyzed with ultraviolet matrix-assisted laser desorption/ionization post-source decay and deuterium exchange.

In an effort to understand the initiating step in metastable-ion decay of UV matrix-assisted laser desorption/ionization (MALDI)-produced ions, we conducted experiments in which we exchanged all active protons for deuterons of tetrameric and hexameric oligodeoxynucleotides. We wish to address the controversial proposal that in the negative-ion mode, as in the positive-ion mode, fragmentation is driven by nucleobase protonation. The results show unambiguously that proton transfer, leading to zwitterion formation, charges a nucleobase prior to its elimination. The zwitterion formation directs fragmentation of both positive and negative oligodeoxynucleotide ions. Poly-T-rich oligodeoxynucleotide tetramers show surprising differences in the negative compared to the positive-ion mode, as thymine is preferentially expelled, instead of a nucleobase with higher proton affinity. For the exceptional case of negatively charged poly-T-rich oligodeoxynucleotide tetramers generated by MALDI, we propose that zwitterion formation with positive charging of a nucleobase leads to base stabilization in the negative-ion mode through an interaction of the positive nucleobase with the excess negative charge. Moreover, backbone cleavages (accompanied by H rearrangement) of a phosophodiester bond give first-generation products that can be traced back to this tripolar complex bearing one positive and two negative charges, all of which may be interacting.

Calibration↗

PubMed2000

Influence of the laser fluence in infrared matrix-assisted laser desorption/ionization with a 2.94 microm Er : YAG laser and a flat-top beam profile.

The dependence of the signal intensity of analyte and matrix ions on laser fluence was investigated for infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry using a flat-top laser beam profile. The beam of an Er : YAG laser (wavelength, 2.94 microm; pulse width, 90 ns) was coupled into a sapphire fiber and the homogeneously illuminated end surface of the fiber imaged on to the sample by a telescope. Three different laser spot sizes of 175, 350 and 700 microm diameter were realized. Threshold fluences of common IR matrices were determined to range from about 1000 to a few thousand J m(-2), depending on the matrix and the size of the irradiated area. In the MALDI-typical fluence range, above the detection threshold ion signals increase strongly with fluence for all matrices, with a dependence similar to that for UV-MALDI. Despite the strongly different absorption coefficients of the tested matrices, varying by more than an order of magnitude at the excitation laser wavelength, threshold fluences for equal spot sizes were found to be comparable within a factor of two. With the additional dependence of fluence on spot size, the deposited energy per volume of matrix at threshold fluence ranged from about 1 kJ mol(-1) for succinic acid to about 100 kJ mol(-1) for glycerol.

Angiotensin I↗

PubMed1999

Matrix-assisted laser desorption/ionization mass spectrometry of DNA using photocleavable biotin.

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.

Base Sequence↗

PubMed1999

Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS.

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 ( T (m)) 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.

Base Sequence↗

PubMed1999

IR-MALDI-mass analysis of electroblotted proteins directly from the membrane: comparison of different membranes, application to on-membrane digestion, and protein identification by database searching.

A systematic membrane study investigating different neutral, cationic derivatized, and hydrophilic PVDF membranes for their suitability to carry out on-membrane tryptic digestions and to obtain infrared-matrix-assisted laser desorption/ionization (IR-MALDI) mass information on the proteolytic fragments directly from the membrane was performed. Clearly, the Immobilon CD membrane (Millipore) showed the most reproducible results over a protein mass range from 12 to 66 kDa. Typical protein load to SDS-PAGE was in the 1-2 micrograms range. The protein amount used for enzymatic treatment was estimated to be in the low picomole range. Now both the intact protein mass and the masses of the specific proteolytic fragments are available directly from the membrane. Protein databases can be searched via search algorithms on the Internet using the information on the intact protein mass and the masses, e.g., of its tryptic fragments. Investigations were performed to search for neutral, enzyme-compatible IR matrixes which allow the enzymatic treatment (on-membrane digestion) while the membrane is matrix-incubated. Thiourea could be tolerated during enzymatic cleavage in solution in concentrations of 15 g/L and resulted in high-quality spectra of intact protein signals and turned, therefore, out to be the most promising candidate.

Algorithms↗

PubMed1999

Detection of double-stranded DNA by IR- and UV-MALDI mass spectrometry.

Double-stranded DNA ranging from 9 kDa to over 500 kDa were desorbed and analyzed by MALDI TOF mass spectrometry. IR-MALDI with glycerol as matrix yielded excellent results for larger double-stranded DNA by adjustment of the ionic strength through the addition of salts. Very little fragmentation and a routine sensitivity in the subpicomole range were observed in IR-MALDI when double-stranded analytes harboring 70 base pairs or more were probed. In the lower mass range (up to approximately 70 base pairs), UV-MALDI with 6-aza-2-thiothymine as matrix was the ionization method of choice because it allowed specific double-stranded complexes containing relatively few base pairs to be desorbed intactly. In this mode, an essentially quantitative detection of the double-stranded form was observed for a 70-mer. The UV-MALDI was accompanied by a significant fragmentation and a resulting reduced sensitivity and mass resolution. The methods described open MALDI-MS for the analysis of large DNA-DNA and DNA-protein complexes.

DNA↗

PubMed1999

Comparative mass spectrometric analyses of Photofrin oligomers by fast atom bombardment mass spectrometry, UV and IR matrix-assisted laser desorption/ionization mass spectrometry, electrospray ionization mass spectrometry and laser desorption/jet-cooling photoionization mass spectrometry.

Photofrin (porfimer sodium) is a porphyrin derivative used in the treatment of a variety of cancers by photodynamic therapy. This oligomer complex and a variety of porphyrin monomers, dimers and trimers were analyzed with five different mass spectral ionization techniques: fast atom bombardment, UV and IR matrix-assisted laser desorption/ionization, electrospray ionization, and laser desorption/jet-cooling photoionization. All five approaches resulted in very similar oligomer distributions with an average oligomer length of 2.7 +/- 0.1 porphyrin units. In addition to the Photofrin analysis, this study provides a side-by-side comparison of the spectra for the five different mass spectrometric techniques.

Dihematoporphyrin Ether↗

PubMed1999

Calcium-induced noncovalently linked tetramers of MRP8 and MRP14 detected by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry.

MRP8 and MRP14 are members of the S100 family of calcium-binding proteins which play an important role during calcium-induced activation of phagocytes. Both proteins form noncovalently associated complexes as a prerequisite for biological functions. The exact stoichiometric composition of these complexes, however, has not been completely clarified yet. In the present study we show for the first time by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI-MS) the calcium-induced formation of noncovalently associated (MRP8/MRP14)2 tetramers. Furthermore, we could determine posttranslational modifications of MRP8 and MRP14, the stoichiometric proportion of the two known MRP14 isoforms in the complexes as well as the number of calcium ions bound to the single MRP8 and MRP14 monomers and tetramers. MRP14 showed a higher affinity for calcium than MRP8. Upon complex formation the calcium binding increased to maximal saturation of the known EF hands in the complexed forms. Calcium-induced stabilization of the MRP8/MRP14 complexes was confirmed by DSC studies. Our results extend scope and application of UV-MALDI-MS by allowing identification of noncovalent protein complexes, the identification of minor alterations of subunits in such complexes as well as the determination of bound calcium ions.

Antigens, Differentiation↗

PubMed1998

Infrared MALDI mass spectrometry of large nucleic acids.

Mass spectrometry has become an increasingly important tool of high accuracy, efficiency, and speed for the routine analysis of nucleic acids. To make it useful for large-scale sequencing of genomic material as required for example in genotyping and clinical diagnosis, it is necessary to find approaches that allow the analysis of sequences much larger than the 100 nucleotides currently possible. Matrix-assisted laser desorption/ionization (MALDI) mass spectra of synthetic DNA, restriction enzyme fragments of plasmid DNA, and RNA transcripts up to a size of 2180 nucleotides are reported. The demonstrated mass accuracy of 1 percent or better and the sample requirement of a few femtomoles or less surpass all currently available techniques for the analysis of large nucleic acids. DNA and RNA can be analyzed with only a limited investment in sample purification.

Calibration↗

PubMed1998

DNA sequence analysis by MALDI mass spectrometry.

Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products. Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination. Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. In the present work the use of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented. The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated by sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.

Animals↗

PubMed1998

Separation and identification of peptides in single neurons by microcolumn liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay analysis.

Microcolumn liquid chromatography (LC) was interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for separation and identification of peptides present in single neurons from the brain of the snail Lymnaea stagnalis. The nanoliter microcolumn LC effluent, mixed off-line with nanoliter matrix solution, was deposited onto the sample target every 60 s, producing fractions of approximately 145 nL in volume, which, upon drying, produced spots of approximately 1 mm in size. At the end of the chromatographic separation, fractions from the sample target were scanned by MALDI-TOF-MS. Identification of peptide peaks was achieved on the basis of LC elution order and mass information. Further identification based on sequence information was carried out for a native peptide fractionated by microcolumn LC from a single neuron with the postsource decay technique.

Animals↗

PubMed1998

Investigations of the metastable decay of DNA under ultraviolet matrix-assisted laser desorption/ionization conditions with post-source-decay analysis and hydrogen/deuterium exchange.

The fragmentation of positive ions of DNA under the conditions of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was investigated by post-source decay (PSD) analysis and hydrogen/deuterium (H/D) exchange. Spectra of five different synthetic 4mer oligonucleotides were recorded. As a main result the hypothesis was confirmed that for these ions all fragment ions result from processes, initiated by protonation/deuteration of a suitable base followed by a loss of this base as a neutral or ion and further backbone cleavages. The three bases adenine, guanine, and cytosine all exhibit comparable lability for fragmentation. The spectra show evidence for an interaction of the adenine base with the phosphate backbone. Signals of fragments containing TT- and CT-cycloadducts were observed in the spectra.

Calibration↗

PubMed1997

Detection of noncovalent tRNA.aminoacyl-tRNA synthetase complexes by matrix-assisted laser desorption/ionization mass spectrometry.

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.

Binding, Competitive↗

PubMed1997

Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling.

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

Amino Acid Sequence↗

PubMed1997

Analysis of proteins by direct-scanning infrared-MALDI mass spectrometry after 2D-PAGE separation and electroblotting.

A novel approach is reported for the analysis and identification of proteins separated by 2D-PAGE with scanning infrared matrix-assisted laser desorption/ionization mass spectrometry (scanning IR-MALDI-MS). The proteins of human blood plasma were separated by 2D-PAGE, electroblotted onto PVDF membranes, incubated in matrix solution, and then scanned by IR-MALDI-MS. Mass contour plots of selected spots were obtained. Protein separation is shown to be conserved by comparison with silver-stained gels. The sensitivity for the protein detection is comparable if not better than that of silver-stained gels. Posttranslational modifications were identified by comparing the measured mass to the one calculated from the known DNA sequence. Adduct formation to unprotected cysteine residues during gel separation is demonstrated for selected proteins.

Blood Proteins↗