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A Tsarbopoulos

Publications and source records attributed to A Tsarbopoulos.

27 records · Page 2Linked to original sources
PubMed1991

Structural analysis of biologically active peptides and recombinant proteins and their modified counterparts by mass spectrometry.

The structural characterization of the Escherichia coli-expressed human interferon alpha-2b (rh-IFN alpha-2b) was carried out by employing the fast atom bombardment (FAB) and plasma desorption (PD) mapping methods. The mass spectral data of the rh-IFN alpha-2b and the trypsin-generated peptide mixture allowed rapid and facile confirmation of the cDNA-derived sequence and determination of the existing disulfide pattern in the protein molecule. The same PD/FAB mapping approach was successfully employed in the structural determination of the iodination reaction product of rh-IFN alpha-2b and the potent vasoconstrictor peptide endothelin.

Amino Acid Sequence↗
PubMed1991

252Cf-plasma desorption and cesium-ion liquid secondary-ion mass spectrometric analysis of recombinant proteins.

The 35 keV Cs+ liquid secondary-ion mass spectrometry (LSIMS) and 252Cf-plasma desorption (PD) mass spectra of recombinant proteins in the 10-25 kDa mass range are compared. Both techniques showed comparable mass accuracy and sensitivity, and in the case of LSIMS, remarkably short analysis time. Analysis by the PD/nitrocellulose method demonstrated slightly higher sensitivity and relatively lower dependence on the salt and buffer content of the protein sample.

Animals↗
PubMed1989

Rapid identification of calbindin-D28k cyanogen bromide peptide fragments by plasma desorption mass spectrometry.

Chicken intestinal calbindin-D28k is an intracellular protein which is believed to have a fundamental role in vitamin D-mediated transport of calcium. A mapping approach based on 252Cf plasma desorption mass spectrometry (PD mapping) was used to screen the DNA-deduced sequence of calbindin-D28k for sequence changes and post-translational modifications. In the PD mapping experiment, purified calbindin-D28k was cleaved with cyanogen bromide and the resulting peptides were subjected to PD mass spectrometric analysis either as a mixture or as high-performance liquid chromatography isolated fractions. The DNA-derived primary structure of calbindin-D28k was confirmed by rapid PD mass spectral identification of the CNBr peptide fragments, and the nature of the N-terminal blocking group was readily determined to be an acetyl group. The relatively non-destructive nature of the PD mass spectrometric analysis allowed the mapping of the N-terminal peptide through an additional in situ V8 protease enzymatic reaction.

Amino Acid Sequence↗
PubMed1988

Demonstration of a 1-3 disulfide bond in a synthetic nonapeptide derived from the signal sequence and N-terminus of human gamma-interferon.

The nonapeptide Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val-Lys was prepared by solid-phase peptide synthesis under oxidizing conditions. Fast atom bombardment mass spectrometric analysis of the untreated molecule produced an ion consistent with a structure involving an intramolecular disulfide bond between Cys(1) and Cys(3). Mass spectra of the peptide after treatment with 2-mercaptoethanol gave signals corresponding to the reduced disulfide form of the peptide and to a mixed disulfide of the peptide with 2-mercaptoethanol. Molecular mechanics calculations of the conformation of the 11-membered ring formed by disulfide bond closure predicted a discrete, low-energy structure resembling the locus of a gamma turn. We hypothesize that this structure may be important in the recognition and cleavage of the signal sequence of the parent molecule.

Chromatography, High Pressure Liquid↗
PubMed1988

Peptide and protein mapping by 252Cf-plasma desorption mass spectrometry.

The mapping of peptide digests by using fast atom bombardment mass spectrometry for evaluating the correctness of known or expected protein sequences is a well-established strategy. A similar approach ("PD mapping") is described which utilizes 252Cf-plasma desorption mass spectrometry (PDMS). This PD mapping approach is demonstrated and evaluated by screening the DNA-deduced sequences of recombinant interleukin-2 and human growth hormone. In the PD mapping experiment, the protein is cleaved either chemically or enzymatically and the molecular weights of the peptides predicted from the proposed amino acid sequence are compared with those determined mass spectrometrically. The relatively nondestructive nature of the PD mass spectrometric analysis allows further confirmation of the sequence assignments of individual peptides through additional steps of enzymatic or chemical modification on the nitrocellulose-bound peptides. The PD mapping method is both fast and sensitive, requiring only low nanomole amounts per map.

Amino Acid Sequence↗
PubMedDate not supplied

Plasma desorption mass spectrometry of natural and recombinant peptides and proteins.

Plasma desorption mass spectrometry (PDMS), which utilizes the fission fragments from the spontaneous decay of californium-252 to ionize large biomolecules, has emerged as a powerful analytical tool in the field of protein chemistry. Because of its high mass range and sensitivity, PDMS is an excellent method for direct molecular weight determination of peptides and small proteins, with much better accuracy than that of the most commonly used classical methods of gel permeation chromatography or SDS-gel electrophoresis. Furthermore, PDMS analysis of the peptide mixture resulting from a specific enzymatic or chemical cleavage of the protein, allows rapid and facile checking of DNA-derived protein sequences and protein structures produced by recombinant DNA technology. The relatively nondestructive nature of the PD mass spectrometric analysis allows further confirmation of the sequence assignments of individual peptide fragments through additional chemical or enzymatic reactions on the PDMS matrix-bound peptides. This PD mapping approach, combined with classical gas phase sequencing, can be used to identify and locate post-translational modifications in proteins, such as glycosylation, phosphorylation, disulfide linkages, and also detect the presence of peptide and protein variants in synthetic, native and recombinant peptides and proteins.

Amino Acid Sequence↗