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F Hillenkamp

Publications and source records attributed to F Hillenkamp.

At least 19 recordsLinked to original sources
PubMed1995

Separation and characterisation of bovine histone H1 subtypes by combined ion-exchange and reversed-phase chromatography and mass spectrometry.

In order to separate and identify histone H1 subtypes from calf thymus we used both electrospray mass spectrometry (ES-MS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) after a three-step chromatographic procedure consisting of reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC) and ion-exchange chromatography (IEC). Under the RP-HPLC conditions described, we obtained two baseline-separated H1-fractions which were characterised by MALDI-TOF-MS. The determined masses ranged from 22,850 to 22,590 for the first fraction and from 22,070 to 21,250 for the second fraction. Further, it was shown that the first fraction contained at least four and the second one at least five subtypes of the histone class H1. Four homogeneous pure H1 subtypes were obtained by a combination of IEC followed by SEC and RP-HPLC. The molecular masses of these four subtypes determined by ES-MS were 22,606, 22,761, 21,347 and 21,263. We obtained six additional molecular masses of histone H1 subtypes from three heterogeneous fractions, namely 22,066, 21,802, 20,586 and 19,817 by ES-MS and 22,800 and 22,675 by MALDI-TOF-MS. The retention times of these fractions and the molecular masses were in agreement with the data obtained from RP-HPLC fractions by MALDI-TOF-MS.

Animals↗
PubMed1995

7-Deaza purine bases offer a higher ion stability in the analysis of DNA by matrix-assisted laser desorption/ionization mass spectrometry.

Oligodeoxynucleotides which contain 7-deaza analogues of the normal purine nucleotides have been synthesized both enzymatically and chemically. When subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis, the modified samples offer both higher stability and increased sensitivity compared to otherwise identical unmodified oligodeoxynucleotides. In view of these observations, models for the fragmentation of oligodeoxynucleotides in MALDI-MS with positive ion detection mode are presented. Additionally, the potential use of 7-deaza purine nucleotides in the MALDI-MS analysis of DNA sequencing reactions is discussed.

Base Sequence↗
PubMed1994

The amino acid sequence of the red kidney bean Fe(III)-Zn(II) purple acid phosphatase. Determination of the amino acid sequence by a combination of matrix-assisted laser desorption/ionization mass spectrometry and automated Edman sequencing.

Purple acid phosphatase of the common bean Phaseolus vulgaris is a homodimeric 110-kDa glycoprotein with a Fe(III)-Zn(II) center in the active site of each monomer. After exchange of Zn(II) for Fe(II), the enzyme spectroscopically and kinetically resembles the mammalian purple acid phosphatases with Fe(III)-Fe(II) centers in monomeric 35-kDa proteins. The kidney bean enzyme consists of 432 amino acids/monomer with five N-glycosylated asparagine residues. The complete amino acid sequence was determined by a combination of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and classical sequencing methods. Our strategy involved mass determination and sequence analysis of all cyanogen-bromide-generated fragments by automated Edman degradation. Limited cleavages with cyanogen bromide were performed to obtain fragments containing still uncleaved Met-Xaa linkages. MALDI mass spectra of these products allowed the characterization of each fragment and the determination of the order of the cyanogen bromide fragments in the intact protein without producing overlapping peptides. For one large 30-kDa methionine-free fragment, the alignment of the Edman-degraded tryptic peptides was obtained by MALDI-MS analysis and enzymic microscale peptide laddering of overlapping Glu-C-generated fragments. The employed strategy shows that the classical method, in combination with modern mass spectrometry, is an attractive approach for primary structure determination in addition to the DNA sequencing method.

Acid Phosphatase↗
PubMed1994

Synthesis and properties of oligodeoxyribonucleotide-polyethylene glycol conjugates.

Pools of oligonucleotide conjugates consisting of 10-400 different molecular species were synthesized. The conjugates contained a varying number of ethylene glycol units attached to 3'-terminal, 5'-terminal and internal positions of the oligonucleotides. Conjugate synthesis was performed by phosphoramidite solid phase chemistry using suitably protected polyethylene glycol phosphoramidites and PEG-derivatized solid supports containing polydisperse PEGs of various molecular weight ranges. The pools were analyzed and fractionated by chromatographic and electrophoretic techniques, and the composition of isolated conjugates was revealed by matrix-assisted laser desorption/ionization mass spectrometry. The number and attachment sites of coupled ethylene glycol units greatly influence the hydrophobicity of the conjugates, as well as their electrophoretic mobilities. Conjugation had little effect on the hybridization behavior of oligonucleotide conjugates with unmodified complementary oligonucleotide strands. Melting temperatures were between 67 and 73 degrees C, depending on the size and number of coupled PEG chains, compared to 68 degrees C for the unmodified duplex. Conjugates with PEG coupled to both 3'- and 5'-terminal positions showed a more than 10-fold increase in exonuclease stability.

Base Sequence↗
PubMed1994

Matrix assisted laser desorption/ionization mass spectrometry of enzymatically synthesized RNA up to 150 kDa.

Enzymatically synthesized RNA samples (in vitro transcripts) were analysed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Spectra of RNA up to 150 kDA (461 nucleotides) are shown. Polymerase generated sample heterogeneity and its contribution to mass resolution are discussed. A time course exonuclease digest of a 55 nt in vitro transcript was analyzed to investigate the performance of MALDI-MS on complex mixtures. Based on these data, the analysis by MALDI-MS of DNA sequencing reactions, produced by the action of an RNA polymerase, is discussed.

Base Sequence↗
PubMed1994

Comparison of IR- and UV-matrix-assisted laser desorption/ionization mass spectrometry of oligodeoxynucleotides.

UV-matrix assisted laser desorption/ionization mass spectrometry (UV-MALDI-MS) with 3-hydroxypicolinic acid as matrix and IR-MALDI-MS with succinic acid as matrix have proved their feasibility for highly accurate and sensitive mass determination of nucleic acids (DNA and RNA). In this work, a detailed comparison of these two MALDI-methods and between positive- and negative ion mass spectra for the analysis of oligodeoxynucleotides is undertaken. Mass spectra of DNA sequences with up to 40 nucleotides are shown. Both linear and reflectron time-of-flight mass analyzers were used within this study and are compared for their potential in the MALDI analysis of oligodeoxynucleotides. The role of molecule-ion fragmentation is also discussed.

Base Sequence↗
PubMed1994

Comparative mapping of recombinant proteins and glycoproteins by plasma desorption and matrix-assisted laser desorption/ionization mass spectrometry.

The mass spectrometric (MS) techniques of 252Cf-plasma desorption (PD) and matrix-assisted laser desorption/ionization (MALDI) are compared in the molecular weight determination and the mapping analysis of several recombinant proteins and glycoproteins. MALDI MS analysis exhibited better sensitivity and mass measurement accuracy and a remarkably short analysis time compared with PD MS analysis. The latter was not successful in the analysis of rhIFN-gamma and the higher mass mammalian cell-derived IL-5 glycoproteins. Mapping of the Escherichia coli-derived rhIFN alpha-2b and rhIL-4 proteins, by direct PD or MALDI MS analysis of the trypsin-generated peptide mixtures provided signals for ca. 95% and 88% of the expected tryptic peptides, respectively. Peptide signals below m/z 1500 were generally more intense in the PD mass spectra, while higher mass signals were more intense in the MALDI mass spectra. Both PD and MALDI MS analyses provided a rapid confirmation of the existing two and three disulfide bonds in the rhIFN alpha-2b and rhIL-4 proteins, respectively. In the mapping of the CHO IL-4 glycoprotein, detection of the trypsin-generated glycopeptides was only possible by MALDI, where their detection was greatly improved by using the super-DHB (sDHB) matrix, a 9:1 mixture of 2,5-dihydroxybenzoic acid (DHB) with 2-hydroxy-5-methoxybenzoic acid. This sDHB matrix also generated significantly enhanced and better resolved MALDI peptide signals, which in turn resulted in a much improved mass measurement accuracy.

Animals↗
PubMed1994

N-terminal variants of fatty acid-binding protein from bovine heart overexpressed in Escherichia coli.

An expression vector for bovine heart fatty acid-binding protein (H-FABP) was constructed by introducing the coding part of the cDNA into the pET-3d vector. Transformed Escherichia coli strain BL21 (DE3)pLysS produced functional recombinant H-FABP up to 40% of the soluble proteins. The expression of fatty acid-binding protein was under the control of the T7-phi 10 promoter and the corresponding T7-RNA-polymerase in turn was induced by isopropyl beta-D-thiogalactopyranoside. By combination of cation exchange chromatography and gel filtration pure recombinant protein was obtained exhibiting isoelectric heterogeneity. Recombinant H-FABP was resolved into at least six variants with isoelectric points between 5.1 and 5.6. After separation by preparative isoelectric focusing the four major variants were digested with trypsin and the resulting peptides were characterized by high performance liquid chromatography (HPLC), matrix assisted laser desorption/ionization (MALDI) mass spectrometry, amino acid sequencing and chemical modification. The structural differences were traced back to the N-termini beginning with either methionine, as expected from the cDNA, or methionine sulfoxide, valine and N-formyl methionine. The latter three arise from oxidation, cleavage of N-terminal methionine and incomplete deformylation, respectively.

Animals↗
PubMed1994

The oligosaccharides of the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean. Determination of the structure by a combination of matrix-assisted laser desorption/ionization mass spectrometry and selective enzymic degradation.

Purple acid phosphatase of the common bean Phaseolus vulgaris (KBPase), a dimeric 110-kDa glycoprotein related to the mammalian purple acid phosphatases with a two-metal cluster at the active site contains five oligosaccharide side chains/monomer. The N-linked glycan structures were characterized by selective enzymic degradation in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The purified protein was cleaved by cyanogen bromide. One 30-kDa large methionine-free fragment required a further tryptic digest. The peptides were separated by HPLC and the glycosylated species were identified both by their heterogeneous mass spectra and by an immunoassay. None of the glycopeptides proved to have more than one glycosylation site. The composition of the carbohydrate moieties were calculated by comparing the mass spectra of the glycopeptides before and after enzymic deglycosylation. These results were complemented by data from a carbohydrate composition analysis. In four of the five peptides an alpha 1-3 fucose attached to the asparagine-linked N-acetylglucosamine prevented removal of the glycan by peptide N-glycosidase F; peptide N-glycosidase A removed all carbohydrates from the peptides. To reveal the sequence of the carbohydrate moiety including the linkage positions between the different saccharides, one of the glycopeptides was degraded by specific exoglycosidases. The enzymic degradations by these hydrolases were monitored by mass spectrometry of small aliquots taken at intervals during the reaction. The detailed structure of this one glycan in conjunction with the respective mass spectra and the composition analysis were used to infer the structure of the other four glycans. All glycans of the KBPase have a complex-type xylose-containing structure with four of the five having an additional fucose.

Acid Phosphatase↗
PubMed1994

Oligosaccharides from human milk as revealed by matrix-assisted laser desorption/ionization mass spectrometry.

In this study neutral and acidic oligosaccharide fractions prepared from human milk have been investigated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The fraction of neutral oligosaccharides was separated by gel permeation chromatography (GPC) and the resulting subfractions were analyzed by MALDI-MS using the positive ion mode. Several low-molecular-weight glycans (degree of polymerization up to 13) were observed whose structures have already been elucidated. In addition, a variety of so far unknown large-sized carbohydrates was detected whose molecular weights range from M(r) 2242 to 8000. The large-sized glycans which possess a low abundance appear to be composed of both lactosamine and fucose residues attached to the lactose unit at the reducing end of the sugar chains with a highly variable stochiometry. Following subfractionation by GPC, acidic (i.e., containing sialic acid) glycans were analyzed by MALDI-MS using both positive and negative ion mode. Because of the inferior stability of acidic glycans, various matrices were applied and compared with respect to signal intensity, resolution, and analyte stability.

Chromatography, Gel↗
PubMed1993

Microphotocoagulation: selective effects of repetitive short laser pulses.

Repetitive exposure to short laser pulses is shown to cause selective damage to absorbing structures (cells, organelles, or enzymes) with pulse energies below the threshold energy for single-pulse damage. Directly adjacent structures are spared in vivo. Additivity of (presumably nonphotochemical) subthreshold effects is demonstrated. Selective damage to the retinal pigment epithelium with sparing of the neural retina is shown (514 nm, 5 microseconds, 1-500 pulses at 500 Hz, 2- to 10-microJ pulse energy). A melanin granule model has been developed and applied to the experimental situation. Histological results as well as the basic mechanism for these effects are discussed.

Animals↗
PubMed1993

Ion stability of nucleic acids in infrared matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with infrared laser light of a wavelength of 2.94 microns has been used for the analysis of nucleic acids. Spectra of oligodeoxynucleotides up to 26 nucleotides, oligothymidylic acids up to 100 nucleotides as well as different synthetic RNA oligomers and RNA transcripts up to 104 nucleotides are presented. A main problem in the analysis of oligodeoxynucleotides was found to be related to the loss of bases. The stability of oligothymidylic acids as opposed to oligodeoxynucleotides containing all four bases indicates that the loss of bases is correlated with A, C and G protonation which decreases the stability of the N-glycosidic bond. Experiments indicate that the breakage of the N-glycosidic bond probably occurs during the desorption process due to proton transfer from the phosphodiester groups to the ionizable bases. RNA displayed a significantly higher stability in MALDI-MS due to the presence of a 2'-OH group. Consequently, signals of RNA transcripts with a length of up to 142 nucleotides could be detected by MALDI-MS. Technical details of the method, including the distribution of positive counterions on the phosphodiester backbone, the upper mass limit and mass accuracy are discussed along with a number of potential analytical applications.

Base Sequence↗
PubMed1993

Matrix-assisted ultraviolet laser desorption/ionization mass spectrometry applied to multiple forms of lipases.

Matrix-assisted ultraviolet laser desorption/ionization mass spectrometry was used to investigate heterogeneous patterns and molecular masses of microbial lipases from Penicillium camembertii, Geotrichum candidum, and Pseudomonas sp. Mass spectral peaks of the native, glycosylated lipases from P. camembertii and G. candidum were broader than those of the corresponding deglycosylated enzymes, indicative of heterogeneous glycosylations. The broader peaks in the mass spectra were caused by an overlapping of unresolved peaks, derived from single glycoprotein species. Molecular masses determined for the deglycosylated proteins were in excellent agreement with those deduced from amino acid composition and sequence data, whereas with conventional biochemical methods (gelfiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis) only very rough estimations of molecular masses were possible. By mass spectrometric analysis of the four fractions of chromatographically separated P. camembertii lipase molecular masses of 29,990, 34,030, 31,990, and 32,140 Da were found before and 29,960, 29,980, 29,990 and 30,010 Da, respectively, after deglycosylation. Thus from the four native fractions of P. camembertii lipase three were glycoproteins. G. candidum lipase showed an average molecular mass of 63,500 Da for the heterogeneously deglycosylated native form and a molecular mass of 59,650 Da for the deglycosylated enzyme. For the Pseudomonas lipase, which could only be isolated with lipids firmly attached, a molecular mass of 32,890 Da was determined, in close agreement with that derived from the cDNA sequence.

Bacterial Proteins↗
PubMed1992

Matrix-assisted laser desorption/ionization mass spectrometry of nucleic acids with wavelengths in the ultraviolet and infrared.

A number of different matrices have been tested and compared for ultraviolet and infrared (UV and IR) matrix-assisted laser desorption/ionization (MALDI) of oligodeoxyribonucleotides and mixtures thereof, as well as ribonucleic acids (tRNA from yeast and rRNA from E. coli). A new technique for removing alkali cations from nucleic acid samples during sample preparation on the sample support is demonstrated. The amount of oligonucleotide sample consumed during a typical measurement in IR-MALDI-MS was determined.

Angiotensin II↗
PubMed1989

Laser radiation tissue interaction.

Mechanisms of interaction between laser radiation and biological tissue are reviewed. Scattering and absorption determine the penetration of radiation into tissue; the heterogeneity of tissues prevents exact model calculations of in-depth energy deposition. The high irradiances, achievable with pulsed lasers, can induce nonlinear absorption, optical breakdown and plasma formation in tissues. Heat conduction and heat convection as well as the build-up and propagation of pressure transients damage tissue outside of the absorbing volume. The limitations to the validity of the first-order rate processes as a description for thermal tissue coagulation and the possibilities for novel photochemistry are discussed.

Animals↗