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M Wilm

Publications and source records attributed to M Wilm.

67 records · Page 4Linked to original sources
PubMed1996

Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.

Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.

Amino Acid Sequence↗
PubMed1996

Parent ion scans of unseparated peptide mixtures.

The nanoelectrospray ion source (NanoES) developed recently has proven to be an excellent tool for the sequencing of peptides out of unseparated mixtures. At lower levels of analyte, however, it is increasingly difficult to distinguish the peptide ions in the spectrum, limiting the overall sensitivity of the procedure. Scans for the parents of common fragmentation products of peptides such as immonium ions allow determination of peptide ion masses even when these ions have signal-to-noise ratios of < 1 in the mass spectrum. With this technique, concentration limits for sequencing peptides could be improved to < 5 fmol/microL in favorable cases. Parent ions scan were used to identify posttranslational modifications of peptides in mixtures, with phosphorylation and glycosylation as examples. Detecting phosphorylation by parent scans increased sensitivity by 1-2 orders of magnitude. Modified peptides can be subjected to tandem mass spectrometry in the same experiment to localize the modification. The present approach expands the range of application of nanoelectrospray and complements established liquid chromatography/mass spectrometry methods.

Amino Acid Sequence↗
PubMed1996

Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.

Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

Amino Acid Sequence↗
PubMed1996

Analytical properties of the nanoelectrospray ion source.

The nanoelectrospray ion source (nanoES) has recently been developed and described theoretically. It is different from conventional electrospray sources and from other miniaturized electrospray sources by (i) its 1-2 microns spraying orifice achieved by pulling the spraying capillary to a fine tip, (ii) its very low flow rate of approximately 20 nL/min and the small size of droplets it generates, and (iii) the absence of solvent pumps and inlet valves. The fabrication and operation of nanoES needles is described in detail. Solutions with up to 0.1 M salt contents could be sprayed without sheath flow or pneumatic assist. Improved desolvation in nanoES led to instrument-limited resolution of the signals of a glycoprotein and the ability to signal average extensively allowed the C-terminal sequencing of a 40 kDa protein. Extensive mass spectrometric and tandem mass spectrometric investigation of the components of an unseparated peptide mixture was demonstrated by verification of 93% of the sequence of carbonic anhydrase. A rapid and robust desalting/concentration step coupled to the nanoES procedure allows the direct analysis of impure samples such as peptide mixtures extracted after in-gel digestion.

Amino Acid Sequence↗
PubMed1996

New carbamate supports for the preparation of 3'-amino-modified oligonucleotides.

A novel approach for the preparation of oligonucleotides carrying amino groups at the 3'-end is described. Several CPG supports having aminoalkyl groups and 3'-amino-2',3'-dideoxynucleosides linked through base-labile carbamate linkages such as 2-(2-nitrophenyl)ethoxycarbonyl and fluorenylmethoxycarbonyl were prepared using two different strategies. These supports are compatible to the standard solid phase phosphite-triester methodology and yield oligonucleotides containing amino groups at the 3'-end. Several properties of the 3'-amino oligonucleotides, such as nuclease resistance, hybridization, and preparation of oligonucleotide conjugates are discussed.

Carbamates↗
PubMed1995

The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins.

The enterotoxin from Clostridium difficile (ToxA) is one of the causative agents of the antibiotic-associated pseudomembranous colitis. In cultured monolayer cells ToxA exhibits cytotoxic activity to induce disassembly of the actin cytoskeleton, which is accompanied by morphological changes. ToxA-induced depolymerization of actin filaments is correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, K. (1995) J. Clin. Invest. 95, 1026-1031). Here we report on the identification of the ToxA-induced modification of Rho. Applying electrospray mass spectrometry, the mass of the modification was determined as 162 Da, which is consistent with the incorporation of a hexose into Rho. From several hexoses tested UDP-glucose selectively served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated peptide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (RhoA, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, and Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [14C]glucose, indicating previous glucosylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA mediates its cytotoxic effects on cells.

Amino Acids↗
PubMed1995

Glucosylation of Rho proteins by Clostridium difficile toxin B.

Toxin A and B, the major virulence factors of Clostridium difficile, are the causative agents of antibiotic-associated pseudomembranous colitis. In cultured cell lines their potent cytotoxicity results from their ability to induce disaggregation of the microfilament cytoskeleton. Toxin B acts on the low-molecular-mass GTPase RhoA, which is involved in the regulation of the actin cytoskeleton. We report here that toxin B catalyses the incorporation of up to one mole of glucose per mole of RhoA at the amino acid threonine at position 37. The modification was identified and localized by tandem electrospray mass spectrometry. UDP-glucose selectively serves as cosubstrate for the monoglucosylation reaction catalysed by toxin B. Microinjection of RhoA previously glucosylated by toxin B into monolayer cells caused disaggregation of actin filaments, indicating a dominant-negative activity of glucosylated RhoA.

Actins↗
PubMed1995

Electrospray mass spectrometry for protein characterization.

Mass spectrometry is a venerable analytical tool that has been used for some time in biochemistry for the analysis of small molecules, such as steroids. More recently, physicists have solved the problems associated with vaporizing and ionizing proteins and peptides, thereby allowing mass spectrometry to take on new roles in investigating protein sequences, structures and modifications.

Mass Spectrometry↗
PubMed1994

Error-tolerant identification of peptides in sequence databases by peptide sequence tags.

We demonstrate a new approach to the identification of mass spectrometrically fragmented peptides. A fragmentation spectrum usually contains a short, easily identifiable series of sequence ions, which yields a partial sequence. This partial sequence divides the peptide into three parts-regions 1, 2, and 3-characterized by the added mass m1 of region 1, the partial sequence of region 2, and the added mass m3 of region 3. We call the construct, m1 partial sequence m3, a "peptide sequence tag" and show that it is a highly specific identifier of the peptide. An algorithm developed here that uses the sequence tag to find the peptide in a sequence database is up to 1 million-fold more discriminating than the partial sequence information alone. Peptides can be identified even in the presence of an unknown posttranslational modification or an amino acid substitution between an entry in the sequence database and the measured peptide. These concepts are demonstrated with model and practical examples of electrospray mass spectrometry/mass spectrometry of tryptic peptides. Just two to three amino acid residues derived by fragmentation are enough to identify these peptides. In peptide mapping applications, even less information is necessary.

Algorithms↗
PubMed1994

[The modification of programmable pacemakers by therapeutic irradiation].

BACKGROUND: More than 300,000 pacemakers are implanted worldwide. During radiation therapy a damage of the pacemaker electronic is possible. METHODS: Twenty pacemakers have been irradiated with photons or electrons experimentally in three different situations: a) pacemaker and pacemaker electrode outside of the irradiation field: b) pacemaker outside, pacemaker electrode inside the irradiation field; c) all things inside the irradiation field. RESULTS: The voltage in the pacemaker electrode produced by the electric field of the accelerator did not exceed 0.8 mV if the electrode was outside the irradiation field. Induced voltage was up to 1.2 mV during irradiation with electrons (18 MeV) and the electrode being inside the treatment field with more than two thirds of its length. After delivering of not more than 10 Gy (photons) to the pacemaker, a decreasing amplitude of the pacemaker pulse occurred. The pulse frequency did not show any deviation. This seems to signal a severe early irreversible damage of the pacemaker that may cause sudden breakdown days or weeks after radiation. Two pacemakers showed a complete breakdown after irradiation with not more than 10 Gy. The others had a complete breakdown beyond doses of 50 Gy. CONCLUSIONS: It is recommended to keep CMOS pacemakers outside the treatment field and to explant if the dose to it was higher than 10 Gy.

Electrons↗
PubMedDate not supplied

Use of mass spectrometric methods for protein identification in receptor research.

In recent years, mass spectrometry has become the method of choice for identifying small amounts of gel separated proteins. Using high mass accuracy peptide mass mapping followed if necessary by nanoelectrospray sequencing, most mammalian proteins can now be identified quickly and sensitively either in amino acid or in EST sequence databases. These methods are illustrated here using an ongoing project in the author's laboratory, a mass spectrometric screen for new mouse brain receptors and their interaction partners.

Animals↗