Biomedical subjects

F Hillenkamp

Publications and source records attributed to F Hillenkamp.

At least 37 records · Page 2Linked to original sources

PubMed1997

Calicheamicin derivatives conjugated to monoclonal antibodies: determination of loading values and distributions by infrared and UV matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization mass spectrometry.

Calicheamicin derivatives (MW approximately 1500) and monoclonal antibodies (MoAbs) conjugated to calicheamicin derivatives (MW approximately 150,000) were analyzed by UV-MALDI/MS, IR-MALDI/MS, and ESI/MS. These materials are potent anticancer agents. Calicheamicin derivatives and conjugates rapidly degrade upon UV irradiation but are relatively stable during IR irradiation and under ESI conditions. A unique feature of IR-MALDI/MS is a 2 times enhancement in resolution relative to UV-MALDI/MS for masses above approximately 50,000 Da resulting in a molecular ion envelope containing a series of partially resolved peaks of the calicheamicin-MoAb conjugates. The mass shift difference between the peak maxima corresponded to the mass change due to the covalent addition of calicheamicin derivatives to the monoclonal antibody. The distribution of the calicheamicin derivatives in the monoclonal antibodies was computed by deconvoluting the partially resolved peak envelope. A unique feature of the ESI mass spectra, under unit resolution conditions, is that the distribution of the carbohydrates can be well resolved for pure MoAbs and can be only partially resolved for conjugated MoAbs. Average loading values for calicheamicia derivatives when conjugated to MoAbs were computed from UV-MALDI/MS, IR-MALDI/MS, and ESI/MS data and the results compared with the average loading values obtained by UV absorption spectrometry. Very low average loading values were computed from UV-MALDI/MS data due to the degradation of the conjugated calicheamicin derivatives during the UV irradiation process. The IR-MALDI/MS average loading values, obtained with glycerol as the matrix, were consistent with the UV absorption spectrometry values for conjugates having hydrolytically stable linkers, but not when the linker contained a hydrolytically labile hydrazone. ESI/MS average loading values were generally lower than the corresponding values obtained by IR-MALDI/MS. The average loading values and distributions obtained using IR-MALDI/MS were more reliable than the corresponding ESI/MS values because the partially resolved, singly and doubly charged peaks in the IR-MALDI spectra can be mathematically deconvoluted, while the overlapping, highly multiply charged peaks of the electrospray spectra can only be partially deconvoluted.

Aminoglycosides↗

PubMed1997

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) of endonuclease digests of RNA.

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 U2digestion. MALDI-MS of the generated fragments is presented as an efficient technique for a direct read out of the nucleotide sequence.

Animals↗

PubMed1997

Matrix-assisted laser desorption/ionisation mass spectrometry of transfer ribonucleic acids isolated from yeast.

tRNATyr 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-trihydroxy-acetophenone 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.

RNA, Fungal↗

PubMed1997

Analysis of fructans from higher plants by matrix-assisted laser desorption/ionization mass spectrometry.

In this communication both matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and high-performance anion-exchange chromatography (HPAEC) have been applied to analyze fructans from higher plants. Size distribution of a commercially available fructan preparation from Dahlia variabilis L. was determined by MALDI-MS. Molecular masses ranged from 2,000 up to 6,000 Da with a peak value of distribution at 2,635 Da. Essentially the same pattern was obtained using HPAEC. Low-molecular-weight fructans from onion bulbs (Allium cepa L.) were studied in more detail. Tissue extracts were analyzed by MALDI-MS without any analyte purification. Mass-spectra of both proteins and oligosaccharides were obtained. For identification, metastable ion scanning was performed. Neither deproteinization nor deionization of the samples affected the oligosaccharide pattern. Using HPAEC, a more complex oligosaccharide pattern was obtained because isomeric glycans were differentiated. However, the overall size distribution was similar to that obtained by MALDI-MS. In further experiments epidermal or parenchyma cell layers of the onion bulb were placed into matrix solution and were then subjected to MALDI-MS and metastable ion scanning as well. By taking this approach, analyte desorption was achieved immediately from plant tissue. Oligosaccharide mass spectra were essentially the same as those of the extracts. To our knowledge, this is the first time that MALDI-MS has been applied as a microprobe to plant tissue. Finally MALDI-MS analysis was performed using single-cell extracts from onion tissues without any purification of the analyte.

Allium↗

PubMed1997

Intracellular degradation of C-peptides in molluscan neurons producing insulin-related hormones.

Single Light Green Cells (LGC) of Lymnaea stagnalis, expressing four genes encoding insulin-related peptides (MIPs) and C-peptides, and sections from the median lip nerve (MLN) were subjected to MALDI-MS. Mass spectra of LGCs and MLNs were almost identical. Masses corresponding to those of the MIPs and some C alpha-peptides could be distinguished. ProMIP III C alpha-peptide and C beta-peptides were not found. The spectra showed additional masses matching those of carboxyterminally truncated C alpha-peptides. Peptides with similar masses were isolated from MLN extracts by HPLC, using electrospray-MS screening. Amino acid sequence analysis revealed intact proMIP I, II and V C alpha-peptides and I, II C alpha-peptide 1-24, 1-22 and 1-15.

Amino Acid Sequence↗

PubMed1997

High-sensitivity peptide mapping by micro-LC with on-line membrane blotting and subsequent detection by scanning-IR-MALDI mass spectrometry.

A novel approach to the on-line mass determination of peptides from digested proteins by scanning infrared matrix-assisted laser desorption/ionization (scanning-IR-MALDI) is described. The peptides were continuously collected directly onto a PVDF (polyvinylidene fluoride) strip during a HPLC run. Individual peptides were detected by lining up the PVDF strip with the UV trace from the HPLC run, using visible dye markers as reference points. The local resolution of the peptides on the PVDF membrane is preserved during matrix incubation for MALDI-MS as shown by comparing the UV chromatogram and the total ion current (TIC) from an on-line coupled electrospray ionization (ESI) mass spectrometer with the scanning-IR-MALDI data from the corresponding areas on the PVDF strip. The intensities of the mass profiles obtained by scanning-IR-MALDI reflect the amount of peptides present on the PVDF strip. The higher sensitivity of IR-MALDI-MS yielded mass information not detectable by ESI-MS. After the scanning-IR-MALDI experiment, the same membrane strip can be used directly for automated Edman degradation. Comparable initial and repetitive yields were obtained for blotted peptides with and without matrix incubation.

Chromatography, High Pressure Liquid↗

PubMed1996

Ice as a matrix for IR-matrix-assisted laser desorption/ionization: mass spectra from a protein single crystal.

Lasers emitting in the ultraviolet wavelength range of 260-360 nm are almost exclusively used for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of macromolecules. Reports about the use of lasers emitting in the infrared first appeared in 1990/1991. In contrast to MALDI in the ultraviolet, a very limited number of reports on IR-MALDI have since been published. Several matrices have been identified for infrared MALDI yielding spectra of a quality comparable to those obtained in the ultraviolet. Water (ice) was recognized early as a potential matrix because of its strong O-H stretching mode near 3 microm. Interest in water as matrix derives primarily from the fact that it is the major constituent of most biological tissues. If functional as matrix, it might allow the in situ analysis of macromolecular constituents in frozen cell sections without extraction or exchanging the water. We present results that show that IR-MALDI of lyophilized proteins, air dried protein solutions, or protein crystals up to a molecular mass of 30 kDa is possible without the addition of any separate matrix. Samples must be frozen to retain a sufficient fraction of the water of hydration in the vacuum. The limited current sensitivity, requiring at least 10 pmol of protein for a successful analysis needs to be further improved.

Animals↗

PubMed1996

A peptidoglycan binding domain in the porin-associated protein (PAP) of Rhodospirillum rubrum FR1.

The porin-associated protein of Rhodospirillum rubrum FR1 was found to contain a peptidoglycan binding motif. A partial fragment of 179 amino acids, obtained by cleavage of PAP with trypsin, Asp-N protease, and CNBr, was sequenced. Substantial sequence homology was found of the C-terminal part (residues 126-179) of porin-associated protein with OmpA, the peptidoglycan-associated lipoprotein of several bacteria, protein F of Pseudomonas aeruginosa, and PIII of Neisseria gonorrhoeae, the latter being also a porin-associated protein. The 179 amino acid fragment comprised about 67% of the mass spectrometrically determined total mass of PAP of 27850 Da.

Amino Acid Sequence↗

PubMed1996

Cell-associated glucans of Burkholderia solanacearum and Xanthomonas campestris pv. citri: a new family of periplasmic glucans.

The cell-associated glucans produced by Burkholderia solanacearum and Xanthomonas campestris pv. citri were isolated by trichloroacetic acid treatment and gel permeation chromatography. The compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry, and high-performance anion-exchange chromatography. B. solanacearum synthesizes only a neutral cyclic glucan containing 13 glucose residues, and X. campestris pv. citri synthesizes a neutral cyclic glucan containing 16 glucose residues. The two glucans were further purified by high-performance anion-exchange chromatography. Methylation analysis revealed that these glucans are linked by 1,2-glycosidic bonds and one 1,6-glycosidic bond. Our 600-MHz homonuclear and 1H-13C heteronuclear nuclear magnetic resonance experiments revealed the presence of a single alpha-1,6-glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The presence of this single alpha-1,6 linkage, however, induces such structural constraints in these cyclic glucans that all individual glucose residues could be distinguished. The different anomeric proton signals allowed complete sequence-specific assignment of both glucans. The structural characteristics of these glucans contrast with those of the previously described osmoregulated periplasmic glucans.

Burkholderia↗

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↗